Trimer Stabilizing HIV Envelope Protein Mutations

ABSTRACT

Human immunodeficiency virus (HIV) envelope proteins having specified mutations that stabilize the trimeric form of the envelope protein are provided. The HIV envelope proteins described herein have an improved percentage of trimer formation and/or an improved trimer yield. Also provided are particles displaying the HIV envelope proteins, nucleic acid molecules and vectors encoding the HIV envelope proteins, as well as compositions containing the HIV envelope proteins, particles, nucleic acid, or vectors.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.17/181,024, filed Feb. 22, 2021, which is a divisional application ofU.S. patent application Ser. No. 16/039,860, filed Jul. 19, 2018, nowU.S. Pat. No. 10,968,254 B2 issued on Apr. 6, 2021, which claimspriority under 35 U.S.C. § 119(b) to European Patent Application No. 17182 075.6, filed Jul. 19, 2017, European Patent Application No. 17 191083.9, filed Sep. 14, 2017, European Patent Application No. 18 158862.5, filed Feb. 27, 2018, and European Patent Application No. 18 178358.0, filed Jun. 18, 2018, the disclosures of which are incorporatedherein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically. The content of the electronic sequence listing(004852-97US3 Sequence Listing.xml; size: 74,021 bytes; and date ofcreation: Jun. 27, 2023) is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus (HIV) affects millions of people worldwide,and the prevention of HIV through an efficacious vaccine remains a veryhigh priority, even in an era of widespread antiretroviral treatment.Antigenic diversity between different strains and clades of the HIVvirus renders it difficult to develop vaccines with broad efficacy.HIV-1 is the most common and pathogenic strain of the virus, with morethan 90% of HIV/AIDS cases deriving from infection with HIV-1 group M.The M group is subdivided further into clades or subtypes, of whichclade C is the largest. An efficacious vaccine ideally would be capableof eliciting both potent cellular responses and broadly neutralizingantibodies capable of neutralizing HIV-1 strains from different clades.

The envelope protein spike (Env) on the HIV surface is composed of atrimer of heterodimers of glycoproteins gp120 and gp41 (FIG. 1A). Theprecursor protein gp160 is cleaved by furin into gp120, which is thehead of the spike and contains the CD4 receptor binding site as well asthe large hypervariable loops (V1 to V5), and gp41, which is themembrane-anchored stem of the envelope protein spike. Like other class Ifusogenic proteins, gp41 contains an N-terminal fusion peptide (FP), aC-terminal transmembrane (TM) domain, and a cytoplasmic domain. Membranefusion between HIV and target cell membranes requires a series ofconformational changes in the envelope protein. HIV vaccines can bedeveloped based upon the envelope protein.

However, various factors make the development of an HIV vaccine basedupon the envelope protein challenging, including the high geneticvariability of HIV-1, the dense carbohydrate coat of the envelopeprotein, and the relatively dynamic and labile nature of the envelopeprotein spike structure. The wild-type envelope protein is unstable dueto its function. Therefore, stabilizing modifications are sometimesintroduced into the envelope structure for generating vaccinecandidates. The envelope protein is a target for neutralizing antibodiesand is highly glycosylated, which reduces the immunogenicity byshielding protein epitopes. All known broadly neutralizing antibodies(bNAbs) do accommodate these glycans.

For vaccine development, it is preferred to use envelope proteins thatcan induce bNAbs. However, most bNAbs only recognize the native envelopeprotein conformation before it undergoes any conformation changes.Therefore, developing a stable envelope protein in its native-likecompact and closed conformation, while minimizing the presentation ofnon-native and thus non-neutralizing epitopes, could improve theefficiency of generating such bNAbs. Previous efforts to produce an HIVvaccine have focused on developing vaccines that contain the pre-fusionectodomain of the trimeric HIV envelope protein, gp140. Gp140 does nothave the transmembrane (TM) and cytoplasmic domains, but unlike gp120,it can form trimer structures. Moreover, these previous efforts havemainly focused on clade A. However, the breadth of the neutralizingantibody response that has been induced is still limited. Therefore, itwould also be beneficial if stabilized native envelope trimers againstmultiple HIV clades were available.

For more than two decades, attempts have been made to develop a stableenvelope protein in its pre-fusion trimer conformation with only limitedsuccess in producing soluble, stable trimers of the envelope proteincapable of inducing a broadly neutralizing antibody response. Forexample, the so-called SOSIP mutations (501C, 605C and 559P) have beenintroduced into the envelope protein sequence to improve the formationof a soluble gp140 trimer fraction (Sanders et al., (2002), 1 Virol.76(17): 8875-89). The so-called SOSIP mutations include cysteineresidues at positions 501 and 605, and a proline residue at position 559according to the numbering in gp160 of HIV-1 isolate HXB2, which is theconventional numbering scheme used in the field. The introduction of thetwo cysteine residues at positions 501 and 605, which are close to oneanother in the three-dimensional protein structure results in adisulfide bridge. SOSIP mutant envelope proteins, such as BG505_SOSIPand B41_SOSIP (envelope proteins from HIV strains BG505 and B41 (i.e.9032-08.A1.4685) strains with SOSIP mutations), have been used invaccine studies and shown to induce tier 2 autologous neutralizing Abs(Sanders et al., Science (2015), 349(6224): 139-140).

However, even though the so-called SOSIP mutations are capable ofstabilizing the trimer form of the envelope protein, the trimer fractionof such SOSIP mutants is usually below 10%, with large amounts ofmonomer and aggregates still produced. Even the SOSIP mutantBG505_SOSIP, which is one of the most promising SOSIP mutant envelopeproteins known to date in terms of its ability to stabilize the trimerform typically yields up to only 25% of the trimer form (Julien et al.,Proc. Nat. Acad. Sci. (2015), 112(38), 11947-52). Moreover, in thistrimer fraction the trimers are not completely stable as they breathe atthe apex. Thus, in addition to the SOSIP mutations, several additionalsubstitutions, such as E64K, A316W, and 201C-433C, have been designed tostabilize the apex and prevent it from breathing (de Taeye et al., Cell(2015), 163(7), 1702-15; Kwon et al., (2015) Nat. Struct. Mol. Biol.22(7) 522-31).

Accordingly, there is a need for stabilized trimers of HIV envelopeproteins that have improved percentage of trimer formation, improvedtrimer yield, and/or improved trimer stability. Preferably, suchstabilized trimers of HIV envelope proteins would also display goodbinding with broadly neutralizing antibodies (bNAbs), and relativelylimited binding to non-broadly neutralizing Abs (non-bNAbs). It is anobject of the invention to provide HIV Env proteins that have improvedtrimer percentages, and preferably also improved trimer yields.

BRIEF SUMMARY OF THE INVENTION

The invention relates to recombinant HIV envelope proteins fromdifferent clades that have improved percentage of trimer formationand/or improved trimer yields as compared to previously described HIVenvelope trimers. Env folding is optimized, strain-specific features arerepaired, and regions of the prefusion-closed conformation important forthe fusion process are stabilized by mutations described herein. Thisprovides a universal approach to optimize the folding and stability ofprefusion-closed HIV-1 envelope trimers. The resulting stable andwell-folded HIV Env trimers are useful for immunization purposes, e.g.to improve chances of inducing broadly neutralizing antibodies andreducing induction of non-neutralizing and weakly neutralizingantibodies upon administration of the recombinant HIV Env trimers. Theinvention also relates to isolated nucleic acid molecules and vectorsencoding the recombinant HIV envelope proteins, cells comprising thesame, and compositions of the recombinant HIV envelope protein, nucleicacid molecule, vector, and/or cells.

In one general aspect, the invention relates to recombinant humanimmunodeficiency virus (HIV) envelope proteins having particular aminoacid residues at identified positions in the envelope protein sequencethat stabilize the formation of trimers.

In certain embodiments, a recombinant HIV envelope (Env) protein of theinvention comprises at position 658 an amino acid chosen from the groupconsisting of Val, Ile, Phe, Met, Ala and Leu, wherein the numbering ofthe positions is according to the numbering in gp160 of HIV-1 isolateHXB2. In certain preferred embodiments, the amino acid at position 658is Val or Ile. In a particularly preferred embodiment, the amino acid atposition 658 is Val.

In certain embodiments, a recombinant HIV envelope (Env) protein of theinvention further comprises one or more of the following amino acidresidues:

-   -   (i) Phe, Leu, Met, or Trp at position 651;    -   (ii) Phe, Ile, Met, or Trp at position 655;    -   (iii) Asn or Gln at position 535;    -   (iv) Val, Ile or Ala at position 589;    -   (v) Phe or Trp at position 573;    -   (vi) Ile at position 204; and/or    -   (vii) Phe, Met, or Ile at position 647,        wherein the numbering of the positions is according to the        numbering in gp160 of HIV-1 isolate HXB2. In certain preferred        embodiments, the indicated amino acid residue at position 651 is        Phe; the indicated amino acid residue at position 655 is Ile;        the indicated amino acid residue at position 535 is Asn; and/or        the indicated amino acid residue at position 573 is Phe.

In certain embodiments, an HIV Env protein of the invention comprisesthe indicated amino acid residues at at least two of the indicatedpositions selected from the group consisting of (i) to (vii) above.

In certain preferred embodiments, a recombinant HIV Env protein of theinvention comprises Val or Ile at position 658 and Ile at position 655.In other preferred embodiment, a recombinant HIV Env protein of theinvention comprises Val or Ile at position 658 and Phe at position 651.In preferred embodiments, the recombinant HIV Env protein of theinvention comprises Ile at position 658 and Ile at position 655. Inother preferred embodiments, the recombinant HIV Env protein of theinvention comprises Ile at position 658 and Phe at position 651. Inother preferred embodiments, the recombinant HIV Env protein of theinvention comprises Ile at position 658, Phe at position 651, and Ile atposition 655. In particularly preferred embodiments, the recombinant HIVEnv protein of the invention comprises Val at position 658 and Ile atposition 655. In other preferred embodiments, the recombinant HIV Envprotein of the invention comprises Val at position 658 and Phe atposition 651. In certain preferred embodiments, the recombinant HIV Envprotein of the invention comprises Val at position 658, Phe at position651, and Ile at position 655.

In certain embodiments, a recombinant HIV Env protein of the inventioncomprises one or more of the mutations described above in a backbone ofan HIV Env consensus amino acid sequence, e.g. from clade C (e.g.comprising the amino acid sequence of SEQ ID NO: 2 or 3) or from clade B(e.g. comprising the amino acid sequence of SEQ ID NO: 4 or 5).

In certain embodiments, a recombinant HIV Env protein of the inventioncomprises one or more of the mutations described above in a parent HIVEnv protein that is a synthetic HIV Env protein, e.g. comprising (a):the amino acid sequence of SEQ ID NO: 6, or (b): SEQ ID NO: 6 with amutation of Glu to Arg at position 166, or (c): (a) or (b) with amutation of the amino acids at positions 501 and 605 into Cys residuesand a mutation of the amino acid at position 559 into a Pro residue, or(d): (a), (b) or (c) having a further furin cleavage site mutation, e.g.replacement of the amino acids at positions 508-511 by RRRRRR (SEQ IDNO: 10), or (e) SEQ ID NO: 7, or (f) a mosaic Env sequence such as Envcomprising the amino acid sequence of SEQ ID NO: 8 or 9.

In certain embodiments, a recombinant HIV Env protein of the inventioncomprises one or more of the mutations described above in a parent HIVEnv protein, which preferably is a wild-type HIV Env protein, preferablyof clade C, comprising at least one repair mutation at an amino acidresidue that is found at the corresponding position at a frequency ofless than 7.5%, preferably less than 2%, of HIV Env sequences in acollection of at least 100, preferably at least 1000, preferably atleast 10000, wild-type HIV Env sequences, wherein the repair mutation isa substitution by an amino acid residue that is found at thecorresponding position at a frequency of at least 10% of HIV Envsequences in said collection and preferably the repair mutation is asubstitution by the amino acid residue that is found at thecorresponding position most frequently in said collection.

In certain preferred embodiments, the HIV Env comprises Val, Ile, Phe,Met, Ala or Leu, preferably Val, at position 658, and: the amino acidresidue at position 651 is Phe; the amino acid residue at position 655is Ile; the amino acid residue at position 535 is Asn; and/or the aminoacid residue at position 573 is Phe.

In certain embodiments, a recombinant HIV Env protein according to theinvention is from a clade C HIV.

In certain embodiments, a recombinant HIV Env protein according to theinvention comprises a HIV Env consensus sequence. In certain embodimentsthereof, the consensus sequence is a clade C HIV Env consensus sequence.In other embodiments, the consensus is a clade B HIV Env consensussequence. In other embodiments, the consensus is a clade A HIV Envconsensus sequence.

In certain preferred embodiments, a recombinant HIV Env protein of theinvention comprises one or more of the mutations described above andfurther comprises Cys at positions 501 and 605, or Pro at position 559,or preferably Cys at positions 501 and 605 and Pro at position 559 (aso-called ‘SOSIP’ variant HIV Env protein), wherein the numbering of thepositions is according to the numbering in gp160 of HIV-1 isolate HXB2.

In certain preferred embodiments, the amino acid residue at position 658is Val; the amino acid residue at position 651 is Phe; the amino acidresidue at position 655 is Ile; the amino acid residue at position 535is Asn; and the amino acid residue at position 573 is Phe.

In certain embodiments, a recombinant HIV Env protein of the inventionfurther comprises one or more of the following amino acid residues:

-   -   (viii) Gln, Glu, Ile, Met, Val, Trp or Phe, preferably Gln or        Glu, at position 588;    -   (ix) Lys at position 64, or Arg at position 66, or Lys at        position 64 and Arg at position 66;    -   (x) Trp at position 316;    -   (xi) Cys at both positions 201 and 433;    -   (xii) Pro at position 556, or Pro at position 558, or Pro at        positions 556 and 558;    -   (xiii) replacement of the loop at amino acid positions 548-568        (HR1-loop) by a loop having 7-10 amino acids, preferably a loop        of 8 amino acids, e.g. having a sequence chosen from any one of        (SEQ ID NOs: 12-17);    -   (xiv) Gly at position 568, or Gly at position 569, or Gly at        position 636, or Gly at both positions 568 and 636, or Gly at        both positions 569 and 636; and/or    -   (xv) Tyr at position 302, or Arg at position 519, or Arg at        position 520, or Tyr at position 302 and Arg at position 519, or        Tyr at position 302 and Arg at position 520, or Tyr at position        302 and Arg at both positions 519 and 520.

In certain embodiments, a recombinant HIV Env protein of the inventionfurther comprises a mutation in the furin cleavage sequence of the HIVEnv protein, such as a replacement at positions 508-511 by RRRRRR (SEQID NO: 10).

In one embodiment, the recombinant HIV Env protein is a gp140 protein.

In another embodiment, the recombinant HIV Env protein is a gp160protein.

In certain embodiments, the recombinant HIV Env protein is truncated inthe cytoplasmic region, e.g. after 7 amino acids of the cytoplasmicregion.

Also disclosed is a method to improve the folding and stability(measured as increased trimer percentage and/or trimer yield) of aparent HIV Env protein, the method comprising repairing the amino acidsequence of the parent HIV Env protein by introducing at least onerepair mutation, preferably at least 3 repair mutations in the parentHIV Env protein, wherein a repair mutation is an amino acid substitutionat an amino acid residue that is present at the corresponding positionat a frequency of less than 7.5%, preferably less than 2%, of HIV Envsequences in a collection of at least 100, preferably at least 500,preferably at least 1000, preferably at least 10000, wild-type HIV Envsequences, wherein the substitution is by an amino acid residue that ispresent at the corresponding position at a frequency of at least 10% ofHIV Env sequences in said collection and preferably the substitution isby the amino acid residue that is present at the corresponding positionmost frequently in said collection. Also disclosed is a repaired HIV Envprotein that is obtainable by said method for improving the folding andstability (measured as increased trimer percentage and/or trimer yield)of a HIV Env protein. Also disclosed is a pharmaceutical compositioncomprising said repaired HIV Env protein. Also disclosed is a method forproducing a HIV Env protein, comprising the method for repairing the HIVEnv protein described herein, and expressing nucleic acid encoding therepaired stabilized HIV Env protein in a recombinant host cell.

Also disclosed is a recombinant HIV Env protein comprising an amino acidsequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 2, wherein position 658, preferably positions 204, 535, 573,589, 647, 651, and 655, and preferably further positions 64, 66, 201,316, 433, 501, 508-511, 556, 558, 559, 588, 548-568 and 605 are nottaken into account when determining the % identity, wherein the aminoacid at position 658 is Val, Ile, Phe, Met, Ala or Leu, preferably Val,and wherein numbering is according to numbering in gp160 of HIV-1isolate HXB2. In certain embodiments thereof, the recombinant HIV Envprotein comprises an amino acid sequence that is at least 98%, 99% or100% identical to SEQ ID NO: 3, wherein position 658, preferablypositions 204, 535, 573, 589, 647, 651, and 655, and preferably furtherpositions 64, 66, 201, 316, 433, 508-511, 556, 558, 588, and 548-568 arenot taken into account when determining the % identity, wherein theamino acid at position 658 is Val, Ile, Phe, Met, Ala or Leu, preferablyVal, and wherein numbering is according to numbering in gp160 of HIV-1isolate HXB2. In another general aspect, the invention relates to arecombinant HIV Env protein comprising an amino acid sequence that is atleast 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4, whereinposition 658, preferably positions 204, 535, 573, 589, 647, 651, and655, and preferably further positions 64, 66, 201, 316, 433, 501,508-511, 556, 558, 559, 588, 548-568 and 605 are not taken into accountwhen determining the % identity, wherein the amino acid at position 658is Val, Ile, Phe, Met, Ala or Leu, preferably Val, and wherein numberingis according to numbering in gp160 of HIV-1 isolate HXB2. In certainembodiments thereof, the recombinant HIV Env protein comprises an aminoacid sequence that is at least 98%, 99% or 100% identical to SEQ ID NO:5, wherein position 658, preferably positions 204, 535, 573, 589, 647,651, and 655, and preferably further positions 64, 66, 201, 316, 433,508-511, 556, 558, 588, and 548-568 are not taken into account whendetermining the % identity, wherein the amino acid at position 658 isVal, Ile, Phe, Met, Ala or Leu, preferably Val, and wherein numbering isaccording to numbering in gp160 of HIV-1 isolate HXB2. In these aspectsand embodiments, one or more of the amino acids at the indicatedpositions that are not taken into account for determining the %identity, are preferably chosen from the amino acids indicated as beingpreferred herein, e.g. Ile at position 204; Phe, Ala, Leu, or Trp atposition 651; etc (see Tables 1 and 2 below).

Also disclosed are a recombinant HIV Env protein comprising an aminoacid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identicalto any one of SEQ ID NOs: 2, 3, 4, 5, 20, 22, 24, 26, 27, 28, 29, 30,31, or 32, wherein SEQ ID NOs: 20, 22, 24, 26, 27, 28, 29, 30, 31, or 32are particularly preferred, and wherein the amino acid at position 658is Val, Ile, Phe, Met, Ala or Leu, preferably Val. In this aspect,preferably positions 204, 535, 573, 589, 647, 651, 655, and 658 andpreferably further positions 64, 66, 201, 316, 433, 508-511, 556, 558,588, and 548-568 are not taken into account when determining the %identity, and wherein numbering is according to numbering in gp160 ofHIV-1 isolate HXB2. Also in this aspect, one or more of the amino acidsat the indicated positions that are not taken into account fordetermining the % identity, are preferably chosen from the amino acidsindicated as being preferred herein in (i)-(vii) of Table 1, (viii)-(xv)of Table 2, and/or (xvi) of Table 1, e.g. Ile at position 204; Phe, Leu,Met, or Trp at position 651; etc.

In another general aspect, the invention relates to a trimeric complexcomprising a noncovalent oligomer of three of any of the recombinant HIVEnv proteins described herein.

In another general aspect, the invention relates to a particle, e.g. aliposome or a nanoparticle, e.g. a self-assembling nanoparticle,displaying on its surface a recombinant HIV Env protein of theinvention.

In another general aspect, the invention relates to an isolated nucleicacid molecule encoding a recombinant HIV Env protein of the inventionand vectors comprising the isolated nucleic acid molecule operablylinked to a promoter. In one embodiment, the vector is a viral vector.In another embodiment, the vector is an expression vector. In onepreferred embodiment, the viral vector is an adenovirus vector.

Another general aspect relates to a host cell comprising the isolatednucleic acid molecule or vector encoding the recombinant HIV Env proteinof the invention. Such host cells can be used for recombinant proteinproduction, recombinant protein expression, or the production of viralparticles.

Another general aspect relates to methods of producing a recombinant HIVEnv protein, comprising growing a host cell comprising an isolatednucleic acid molecule or vector encoding the recombinant HIV Env proteinof the invention under conditions suitable for production of therecombinant HIV Env protein.

Yet another general aspect relates to a composition comprising arecombinant HIV Env protein, trimeric complex, isolated nucleic acidmolecule, or vector as described herein, and a pharmaceuticallyacceptable carrier.

In another general aspect, the invention relates to a method ofimproving the trimer formation of an HIV Env protein, the methodcomprising introducing the substitution of the amino acid (e.g. Lys) atposition 658 by Val, Ile, Phe, Met, Ala, or Leu, preferably by Val, intoa parent HIV Env protein, wherein the numbering of the positions isaccording to the numbering in gp160 of HIV-1 isolate HXB2.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood that the invention is notlimited to the precise embodiments shown in the drawings.

In the figures:

FIGS. 1A and 1B show a schematic representation of the structure of HIVenvelope (Env) proteins;

FIG. 1A shows a full length HIV Env protein; and

FIG. 1B shows a soluble HIV Env protein containing the so-called SOSIPmutations and a C-terminal truncation beginning at residue 664 accordingto the numbering in gp160 of HIV-1 isolate HXB2 (SOSIP.644 sequence);

FIGS. 2A and 2B show the percentage of trimer formation (FIG. 2A) andthe trimer yield (FIG. 2B) for recombinant HIV Env proteins with certainmutations as measured by AlphaLISA assay as described in Example 3; therecombinant HIV Env proteins tested contained a single, double, ortriple amino acid substitution introduced into the backbone HIV Envconsensus clade C sequence ConC_SOSIP (SEQ ID NO: 3); trimer percentageand trimer yield were determined based on the binding of the trimerspecific monoclonal antibody (mAb) PGT145 to each of the recombinant HIVEnv proteins; the trimer yield and percentage of trimer formation foreach of the recombinant HIV Env proteins of the invention is compared tothat of an envelope protein having the backbone ConC_SOSIP sequencewithout any additional trimer stabilizing mutations described herein;

FIG. 3 shows the chromatograms from size exclusion chromatography withmulti-angle light scattering (SEC-MALS) analysis of recombinant HIV Envproteins with certain mutations; the recombinant HIV Env proteins testedcontained a single amino acid substitution introduced into the backboneHIV Env consensus clade C sequence ConC_SOSIP (SEQ ID NO: 3), and werepurified by lectin affinity chromatography as described in Example 2;SEC-MALS analysis was performed as described in Example 3; the peakcorresponding to the trimer form is indicated in each of thechromatograms;

FIGS. 4A-4B show the percentage of trimer formation (FIG. 4A) and thetrimer yield (FIG. 4B) for recombinant HIV Env proteins having a singleamino acid substitution introduced into the backbone HIV Env consensusclade B sequence ConB_SOSIP (SEQ ID NO: 5) with certain mutationscompared to that of the envelope protein having the backbone ConB_SOSIPsequence without any additional trimer stabilizing mutations asdescribed in Example 4; trimer yield and the percentage of trimerformation was measured by AlphaLISA assay;

FIGS. 5A-5B show the percentage of trimer formation and trimer yield forrecombinant HIV Env proteins having amino acid substitutions introducedinto the backbone synthetic HIV envelope protein sequenceDS_sC4_SOSIP_E166R as described in Example 5; the percentage of trimerformation and trimer yield were measured by AlphaLISA assay.

FIG. 6 shows the chromatograms from size exclusion chromatography withmulti-angle light scattering (SEC-MALS) analysis of recombinant HIV Envproteins with certain mutations; the recombinant HIV Env proteins testedcontained the single K655I mutation with in each next variant anadditional mutation introduced into the backbone HIV Env consensus cladeC sequence ConC_SOSIP (SEQ ID NO: 3), and were purified by lectinaffinity chromatography as described in example 2; SEC-MALS analysis wasperformed as described in example 3; the peak representing the gp140monomers in ConC_SOSIP are indicated by a shaded box, at the right sideof the trimer peak. The lower panel shows a zoom in on the lower part ofthe graph, such that it can be seen that each additional mutation causesa further drop in the height of the gp140 monomer peak.

FIGS. 7A-7D show the percentage of trimer formation (FIG. 7A, B fordifferent experiments) and the trimer yield (FIG. 7C, D for differentexperiments) for recombinant HIV Env proteins with the indicatedmutations as described in Example 8, measured by AlphaLISA assay.

FIG. 8 shows the SEC-MALS chromatograms of recombinant HIV Env proteinswith the indicated mutations, as described in Example 8.

FIGS. 9A-9B show the percentage of trimer formation (FIG. 9A) and thetrimer yield (FIG. 9B) for BG505_SOSIP (derived from a wild-type clade Astrain) having single amino acid substitutions and combinations ofsubstitutions compared to that of the envelope protein having thebackbone BG505_SOSIP sequence without any additional trimer stabilizingmutations as described in Example 9. Trimer yield and the percentage oftrimer formation was measured by AlphaLISA assay.

FIG. 10 shows the chromatograms from size exclusion chromatography withmulti-angle light scattering (SEC-MALS) analysis of recombinant HIV Envproteins; SEC-MALS analysis was performed on culture supernatant of Envtransfected cells. The peak corresponding to the trimer form elutesbetween 7 and 7.5 minutes. The dark grey line is BG505_SOSIP (derivedfrom a wild-type clade A strain) and the light grey line is BG505_SOSIPwith L556P, K655I, M535N, N651F, D589V, K588E substitutions.

FIGS. 11A-11B show the trimer yield for C97ZA_SOSIP variants, describedin Example 10. Trimer yield of C97ZA with three stabilizingsubstitutions (L556P, T651F and M535N) (FIGS. 10A and B). In FIG. 11B,the Env sequence was further optimized by additional mutations (21 extramuts) that were added to repair the C97ZA Env sequence according to theconceptual framework described in FIG. 13 and by introduction ofadditional stabilizing substitutions (K655I, D589V, A204I and K588E).Trimer yield and the percentage of trimer formation were measured byAlphaLISA assay. The signals were normalized to ConC_SOSIP signal thatwas set at 1. PNGS is potential N-glycosylation site.

FIG. 12 . Trimer yield of HIV-1 Env strain DU422 with four stabilizingsubstitutions (see Example 11 for details). All numbers were normalizedto ConC_SOSIP (not shown) which was set to 1.

FIG. 13 . Universal concept for repairing HIV-1 Env sequence illustratedfor strain C97ZA. The residue with the highest frequency of occurrence(referred to herein as ‘consensus residue’) in the total HIV-1 database(top bars) and strain C97ZA residue (bottom bars) sorted from low tohigh occurrence percentage of C97ZA residue position. C97ZA sequencepositions to be substituted to consensus residue were selected based onthe following criteria: Positions with a C97ZA residue that occurs lessthan 2% in Env database sequences (black bars). Positions with a C97ZAresidue that occur between 2% and 7.5% in Env database sequences and areburied or partly buried (dark grey bars). Positions that are exposed andhydrophobic in C97ZA and hydrophilic consensus residues (two lightestgrey bars) and a position that is a potential N-glycosylation site(PNGS) consensus residue (S234N).

FIG. 14 . Prefusion closed HIV ENV_SOSIP trimers through sequence repairand mutational stabilization. AlphaLISA signals in cell culturesupernatant for all SOSIP variants normalized to the ConC_SOSIP forbroadly neutralizing antibodies. For respective HIV Env variants,“stabilized” is indicated by ‘STAB’ and “repaired” is indicated by‘REP’.

FIG. 15 . Analytical SEC profile of control Env_SOSIP variants (BackboneSOSIP), repaired Env variants according to the concept described inExample 12 and FIG. 13 , and Env variants with additional stabilizingsubstitutions according to table 3 using cell culture supernatants aftertransfection. Mock signal of cell culture supernatant was subtractedfrom all profiles. The trimer peaks are indicated with *.

FIG. 16 . Trimer yield of HIV-1 Env ConC variants without thestabilizing SOSIP modifications.

FIGS. 17A-17B. Trimer yield (A) and trimer percentage (B) of ConC_SOSIPwith mutations at positions 589, 647, 651 and 655 to methionines. Allnumbers were normalized to ConC_SOSIP (not shown) which was set to 1. Anerror bar is shown at the right end of the bars.

FIG. 18 . Analytical SEC profiles of ConB_SOSIP and ConB_SOSIP_Q658V,using cell culture supernatants after transfection. Mock signal of cellculture supernatant was subtracted from all profiles. The trimer peaksare indicated with a line and labeled with trimer, and the monomer peakis labeled with monomer.

FIG. 19 . Serum ID50 titers in rabbits. Serum ID50 titers in rabbits(one animal per line in heatmap) after priming with ConC_SOSIP Ni-NTAliposomes (stabilized ConC_SOSIP.v3, SEQ ID NO: 28), and 3 boostimmunizations with Env proteins (repaired and stabilized (RAS)sC4_SOSIP.v4, SEQ ID NO: 32; RAS C97ZA_SOSIP.v2, SEQ ID NO: 30; RASDu422_SOSIP.v1, SEQ ID NO: 31) covalently coupled to liposomes(Env-liposomes) as described in example 15. Control animals (n=2) wereinjected with Tris buffer. Using a 7-virus clade C Tier 2 panel (columnsin heatmap with clade C Env isolate code at the bottom).

FIG. 20 . Effect of stabilizing mutations in membrane-bound Consensus CSOSIP Env. FACS experiment comparing integrated MFI of membrane boundConC_SOSIP_FL with stabilized ConC_SOSIP_FL. Data are plotted as meanfold-change to the ConC_SOSIP_FL backbone±SD (duplicate staining). Fordetails see example 18.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification. All patents,published patent applications and publications cited herein areincorporated by reference as if set forth fully herein. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

Unless otherwise stated, any numerical values, such as a concentrationor a concentration range described herein, are to be understood as beingmodified in all instances by the term “about.” Thus, a numerical valuetypically includes ±10% of the recited value. As used herein, the use ofa numerical range expressly includes all possible subranges, allindividual numerical values within that range, including integers withinsuch ranges and fractions of the values unless the context clearlyindicates otherwise.

Amino acids are referenced throughout the disclosure. There are twentynaturally occurring amino acids, as well as many non-naturally occurringamino acids. Each known amino acid, including both natural andnon-natural amino acids, has a full name, an abbreviated one lettercode, and an abbreviated three letter code, all of which are well knownto those of ordinary skill in the art. For example, the three and oneletter abbreviated codes used for the twenty naturally occurring aminoacids are as follows: alanine (Ala; A), arginine (Arg; R), aspartic acid(Asp; D), asparagine (Asn; N), cysteine (Cys; C), glycine (Gly; G),glutamic acid (Glu; E), glutamine (Gln; Q), histidine (His; H),isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met;M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).Amino acids can be referred to by their full name, one letterabbreviated code, or three letter abbreviated code.

Unless the context clearly dictates otherwise, the numbering ofpositions in the amino acid sequence of an HIV envelope protein as usedherein is according to the numbering in gp160 of HIV-1 isolate HXB2 asfor instance set forth in Korber et al. (Human Retroviruses and AIDS1998: A Compilation and Analysis of Nucleic Acid and Amino AcidSequences. Korber et al., Eds. Theoretical Biology and Biophysics Group,Los Alamos National Laboratory, Los Alamos, N. Mex.), which isincorporated by reference herein in its entirety. Numbering according toHXB2 is conventional in the field of HIV Env proteins. The gp160 ofHIV-1 isolate HXB2 has the amino acid sequence shown in SEQ ID NO: 1.Alignment of an HIV Env sequence of interest with this sequence can beused to find the corresponding amino acid numbering in the sequence ofinterest.

The term “percent (%) sequence identity” or “% identity” describes thenumber of matches (“hits”) of identical amino acids of two or morealigned amino acid sequences as compared to the number of amino acidresidues making up the overall length of the amino acid sequences. Inother terms, using an alignment, for two or more sequences thepercentage of amino acid residues that are the same (e.g. 95%, 97% or98% identity) may be determined, when the sequences are compared andaligned for maximum correspondence as measured using a sequencecomparison algorithm as known in the art, or when manually aligned andvisually inspected. The sequences which are compared to determinesequence identity may thus differ by substitution(s), addition(s) ordeletion(s) of amino acids. Suitable programs for aligning proteinsequences are known to the skilled person. The percentage sequenceidentity of protein sequences can, for example, be determined withprograms such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g using theNCBI BLAST algorithm (Altschul S F, et al (1997), Nucleic Acids Res.25:3389-3402).

A ‘collection of HIV Env sequences’ as used herein is a collection of arepresentative number (e.g. at least 100, or 500, or 1000, or more) ofrandom sequences of wild-type HIV Env proteins, which may be from thesame clade (e.g. clade C) or from different clades (e.g. clades A, B, C,etc). Suitable collections of such sequences are available in databases,or subcollections can be extracted therefrom, e.g. the HIV SequenceDatabase (Los Alamos National Laboratory). Such a collection comprisespreferably at least 100 HIV Env protein sequences, 1000 HIV Env proteinsequences, at least 10000 HIV Env protein sequences, at least 50000 HIVEnv protein sequences, and may contain more than 90000 HIV Env proteinsequences.

A ‘corresponding position’ in a HIV Env protein refers to position ofthe amino acid residue when at least two HIV Env sequences are aligned.Unless otherwise indicated, amino acid position numbering for thesepurposes is according to numbering in gp160 of HIV-1 isolate HXB2, ascustomary in the field.

A ‘stabilizing mutation’ as used herein is a mutation as describedherein in any of entries (i)-(vii), or (xvi), of Table 1, or (viii)-(xv)of Table 2, which increases the percentage of trimer and/or the trimeryield (which can for instance be measured according to AlphaLISA orSEC-MALS assays described herein) of an HIV Env protein as compared to aparent molecule when the mutation is introduced by substitution of thecorresponding amino acid in said parent molecule. The amino acidsresulting from such stabilizing mutations typically are rarely, if atall, found in Env proteins of wild-type HIV isolates.

A ‘repair mutation’ as used herein is a substitution of an amino acidresidue in a parent HIV Env protein, which amino acid residue is presentin less than 7.5%, preferably less than 2%, at the correspondingposition in a collection of HIV Env protein sequences, wherein thesubstitution is by an amino acid that is present at the correspondingposition in said collection more frequently, e.g. in at least 10% of HIVEnv proteins in said collection, and preferably is by an amino acid thatis present at the corresponding position in said collection in at least20% of HIV Env proteins or is the most frequently occurring amino acidat the corresponding position in said collection. The amino acidsresulting from such repair mutations thus typically are found in arelatively high percentage of Env proteins of wild type HIV isolates,and may in several cases be the same as those at the correspondingposition in consensus HIV Env sequences.

A ‘repaired and stabilized’ HIV Env sequence as used herein typicallycontains at least one repair mutation and at least one stabilizingmutation, preferably multiple repair mutations and multiple stabilizingmutations as compared to the parent HIV Env sequence.

The terms ‘natural’ or ‘wild-type’ are used interchangeably herein whenreferring to HIV strains (or Env proteins therefrom), and refer to HIVstrains (or Env proteins therefrom) as occurring in nature, e.g. such asin HIV-infected patients.

The invention generally relates to recombinant HIV envelope (Env)proteins comprising certain amino acid substitutions at indicatedpositions in the envelope protein sequence that stabilize the trimerform of the envelope protein. Introducing one or more of the identifiedamino acid substitutions of the invention into the sequence of an HIVenvelope protein can result in an increased percentage of trimerformation and/or an increased trimer yield. This can for instance bemeasured using trimer-specific antibodies, melting temperature, sizeexclusion chromatography, and binding to antibodies that bind tocorrectly folded (stable trimeric) or alternatively to incorrectlyfolded (non-stable or non-trimeric) Env protein, and increased trimerpercentage and/or trimer yield is considered indicative of stable,native, correctly folded Env protein.

Human immunodeficiency virus (HIV) is a member of the genusLentivirinae, which is part of the family of Retroviridae. Two speciesof HIV infect humans: HIV-1 and HIV-2. HIV-1 is the most common strainof HIV virus, and is known to be more pathogenic than HIV-2. As usedherein, the terms “human immunodeficiency virus” and “HIV” refer to, butare not limited to, HIV-1 and HIV-2. In preferred embodiments, HIVrefers to HIV-1.

HIV is categorized into multiple clades with a high degree of geneticdivergence. As used herein, the term “HIV clade” or “HIV subtype” refersto related human immunodeficiency viruses classified according to theirdegree of genetic similarity. The largest group of HIV-1 isolates iscalled Group M (major strains) and consists of at least ten clades, Athrough J.

In one general aspect, the invention relates to a recombinant HIVenvelope (Env) protein. The term “recombinant” when used with referenceto a protein refers to a protein that is produced by a recombinanttechnique or by chemical synthesis in vitro. According to embodiments ofthe invention, a “recombinant” protein has an artificial amino acidsequence in that it contains at least one sequence element (e.g., aminoacid substitution, deletion, addition, sequence replacement, etc.) thatis not found in the corresponding naturally occurring sequence.Preferably, a “recombinant” protein is a non-naturally occurring HIVenvelope protein that is optimized to induce an immune response orproduce an immunity against one or more naturally occurring HIV strains.

The terms “HIV envelope protein,” “HIV Env,” and “HIV Env protein” referto a protein, or a fragment or derivative thereof, that is in natureexpressed on the envelope of the HIV virion and enables an HIV to targetand attach to the plasma membrane of HIV infected cells. The terms“envelope” and “Env” are used interchangeably throughout the disclosure.The HIV env gene encodes the precursor protein gp160, which isproteolytically cleaved into the two mature envelope glycoproteins gp120and gp41. The cleavage reaction is mediated by a host cell protease,furin (or by furin-like proteases), at a sequence motif highly conservedin retroviral envelope glycoprotein precursors. More specifically, gp160trimerizes to (gp160)₃ and then undergoes cleavage into the twononcovalently associated mature glycoproteins gp120 and gp41. Viralentry is subsequently mediated by a trimer of gp120/gp41 heterodimers.Gp120 is the receptor binding fragment, and binds to the CD4 receptor(and the co-receptor) on a target cell that has such a receptor, suchas, e.g., a T-helper cell. Gp41, which is non-covalently bound to gp120,is the fusion fragment and provides the second step by which HIV entersthe cell. Gp41 is originally buried within the viral envelope, but whengp120 binds to a CD4 receptor and co-receptor, gp120 changes itsconformation causing gp41 to become exposed, where it can assist infusion with the host cell. Gp140 is the ectodomain of gp160.

According to embodiments of the invention, an “HIV envelope (Env)protein” can be a gp160 or gp140 protein, or combinations, fusions,truncations, or derivatives thereof. For example, an “HIV envelopeprotein” can include a gp120 protein noncovalently associated with agp41 protein. An “HIV envelope protein” can also be a truncated HIVenvelope protein including, but not limited to, envelope proteinscomprising a C-terminal truncation in the ectodomain (i.e. the domainthat extends into the extracellular space), a truncation in the gp41,such as a truncation in the ectodomain of gp41, in the transmembranedomain of gp41, or a truncation in the cytoplasmic domain of gp41. AnHIV envelope protein can also be a gp140, corresponding to the gp160ectodomain, or an extended or truncated version of gp140. Expression ofgp140 proteins has been described in several publications (e.g. Zhang etal., 2001; Sanders et al., 2002; Harris et al., 2011), and the proteincan also be ordered from service providers, in different variants e.g.based on different HIV strains. A gp140 protein according to theinvention can have a cleavage site mutation so that the gp120 domain andgp41 ectodomain are not cleaved and covalently linked, or alternativelythe gp120 domain and gp41 ectodomain can be cleaved and covalentlylinked, e.g. by a disulfide bridge (such as for instance in the SOSIPvariants). An “HIV envelope protein” can further be a derivative of anaturally occurring HIV envelope protein having sequence mutations,e.g., in the furin cleavage sites, and/or so-called SOSIP mutations. AnHIV envelope protein according to the invention can also have a cleavagesite so that the gp120 and gp41 ectodomain can be non-covalently linked.

In preferred embodiments of the invention, the HIV Env protein is agp140 protein or a gp160 protein, and more preferably a gp140 protein.In other preferred embodiments the Env protein is truncated, e.g. bydeletion of the residues after the 7th residue of the cytoplasmic regionas compared to a natural Env protein.

According to embodiments of the invention, an “HIV envelope protein” canbe a trimer or a monomer, and is preferably a trimer. The trimer can bea homotrimer (e.g., trimers comprising three identical polypeptideunits) or a heterotrimer (e.g., trimers comprising three polypeptideunits that are not all identical). Preferably, the trimer is ahomotrimer. In case of a cleaved gp140 or gp160, it is a trimer ofpolypeptide units that are gp120-gp41 dimers, and in case all three ofthese dimers are the same, this is considered a homotrimer.

An “HIV envelope protein” can be a soluble protein, or a membrane boundprotein. Membrane bound envelope proteins typically comprise atransmembrane domain, such as in the full length HIV envelope proteincomprising a transmembrane domain (TM) as shown in FIG. 1A. Membranebound proteins can have a cytoplasmic domain, but do not require acytoplasmic domain to be membrane bound. Soluble envelope proteinscomprise at least a partial or a complete deletion of the transmembranedomain. For instance, the C-terminal end of a full length HIV envelopeprotein can be truncated to delete the transmembrane domain, therebyproducing a soluble protein, as shown in FIG. 1B. However, the HIVenvelope protein can still be soluble with shorter truncations andalternative truncation positions to those shown in FIG. 1B. Truncationcan be done at various positions, and non-limiting examples are afteramino acid 664, 655, 683, etc. which all result in soluble protein. Amembrane-bound Env protein according to the invention may comprise acomplete or a partial C-terminal domain (e.g. by partial deletion of theC-terminal cytoplasmic domain, e.g. in certain embodiments after the7^(th) residue of the cytoplasmic region) as compared to a native Envprotein.

A signal peptide is typically present at the N-terminus of the HIV Envprotein when expressed, but is cleaved off by signal peptidase and thusis not present in the mature protein. The signal peptide can beinterchanged with other signal sequences, and two non-limiting examplesof signal peptides are provided herein in SEQ ID NOs: 11, 18, 33, and34.

According to embodiments of the invention, the HIV envelope protein,e.g., gp160, or gp140, can be derived from an HIV envelope proteinsequence from any HIV clade (or ‘subtype’), e.g., clade A, clade B,clade C, clade D, clade E, clade F, clade G, clade H, etc, orcombinations thereof (such as in ‘circulating recombinant forms’ or CRFsderived from recombination between viruses of different subtypes, e.gBC, AE, AG, BE, BF, ADG, etc). The HIV envelope protein sequence can bea naturally occurring sequence, a mosaic sequence, a consensus sequence,a synthetic sequence, or any derivative or fragment thereof. A “mosaicsequence” contains multiple epitopes derived from at least three HIVenvelope sequences of one or more HIV clades, and may be designed byalgorithms that optimize the coverage of T-cell epitopes. Examples ofsequences of mosaic HIV envelope proteins include those described in,e.g., Barouch et al, Nat Med 2010, 16: 319-323; and WO 2010/059732, suchas for instance those shown in SEQ ID NOs: 8 and 9. As used herein“consensus sequence” means an artificial sequence of amino acids basedon an alignment of amino acid sequences of homologous proteins, e.g. asdetermined by an alignment (e.g. using Clustal Omega) of amino acidsequences of homologous proteins. It is the calculated order of mostfrequent amino acid residues, found at each position in a sequencealignment, based upon sequences of Env from at least 1000 natural HIVisolates. A “synthetic sequence” is a non-naturally occurring HIVenvelope protein that is optimized to induce an immune response orproduce immunity against more than one naturally occurring HIV strains.Mosaic HIV envelope proteins are non-limiting examples of synthetic HIVenvelope proteins. In preferred embodiments of the invention, the parentHIV Env protein is a consensus Env protein, or a synthetic Env protein.In the parent Env protein, a mutation is introduced to result in aminoacid Val, Ile, Phe, Met, Ala, or Leu, preferably Val or Ile, at position658. Optionally, such HIV Env protein may further have at least one ofthe indicated amino acids at the indicated positions (i)-(vii) describedherein in Table 1. Particularly preferred are consensus Env proteinshaving at least one, preferably at least two of the indicated amino acidresidues at the indicated positions (i)-(vii), preferably having furtherSOSIP and/or furin cleavage site mutations as described below.

In certain embodiments of the invention, an HIV envelope protein,whether a naturally occurring sequence, mosaic sequence, consensussequence, synthetic sequence etc., comprises additional sequencemutations e.g., in the furin cleavage sites, and/or so-called SOSIPmutations.

In some embodiments of the invention, an HIV envelope protein of theinvention has further mutations and is a “SOSIP mutant HIV Env protein.”The so-called SOSIP mutations are trimer stabilizing mutations thatinclude the ‘SOS mutations’ (Cys residues at positions 501 and 605,which results in the introduction of a possible disulfide bridge betweenthe newly created cysteine residues) and the ‘IP mutation’ (Pro residueat position 559). According to embodiments of the invention, a SOSIPmutant Env protein comprises at least one mutation selected from thegroup consisting of Cys at positions 501 and 605; Pro at position 559;and preferably Cys at positions 501 and 605 and Pro at position 559. ASOSIP mutant HIV Env protein can further comprise other sequencemutations, e.g., in the furin cleavage site. In addition, in certainembodiments it is possible to further add mutations such that the Envprotein comprises Pro at position 556 or position 558 or at positions556 and 558, which were found to be capable of acting not only asalternatives to Pro at position 559 in a SOSIP variant, but also asadditional mutations that could further improve trimer formation of aSOSIP variant that already has Pro at position 559.

In certain preferred embodiments of the invention, a SOSIP mutant HIVEnv protein comprises Cys at positions 501 and 605, and Pro at position559.

In certain embodiments, an HIV envelope protein of the invention furthercomprises a mutation in the furin cleavage site. The mutation in thefurin cleavage sequence can be an amino acid substitution, deletion,insertion, or replacement of one sequence with another, or replacementwith a linker amino acid sequence. Preferably in the present invention,mutating the furin cleavage site can be used to optimize the cleavagesite, so that furin cleavage is improved over wild-type, for instance bya replacement of the sequence at residues 508-511 with RRRRRR (SEQ IDNO: 10) [i.e. replacement of a typical amino acid sequence (e.g. EK) atpositions 509-510 with four arginine residues (i.e. two replacements andtwo additions), while at positions 508 and 511, there are alreadyarginine residues present in most HIV Env proteins, so these typicallydo not need to be replaced, but since the end result in literature isoften referred to as amino acid sequence RRRRRR, we kept thisnomenclature herein]. Other mutations that improve furin-cleavage areknown and can also be used. Alternatively, it is possible to replace thefurin cleavage site with a linker, so that furin cleavage is no longernecessary but the protein will adopt a native-like conformation (e.g.described in (Sharma et al, 2015) and (Georgiev et al, 2015)).

In particular embodiments of the invention, an HIV envelope protein ofthe invention further comprises both the so-called SOSIP mutations(preferably Cys at positions 501 and 605, and Pro at position 559) and asequence mutation in the furin cleavage site, preferably a replacementof the sequence at residues 508-511 with RRRRRR (SEQ ID NO: 10). Incertain preferred embodiments, the HIV Env comprises both the indicatedSOSIP and furin cleavage site mutations, and in addition furthercomprises a Pro residue at position 556 or 558, most preferably at bothpositions 556 and 558.

In preferred embodiments of the invention, the amino acid sequence ofthe HIV envelope protein is a consensus sequence, such as an HIVenvelope clade C consensus or an HIV envelope clade B consensus. In aparticularly preferred embodiment, the amino acid sequence of the HIVenvelope protein is an HIV envelope clade C consensus.

Exemplary HIV envelope proteins that can be used in the inventioninclude HIV envelope clade C consensus (SEQ ID NO: 2) and HIV envelopeclade B consensus (SEQ ID NO: 4). These HIV envelope clade C and clade Bconsensus sequences can comprise additional mutations that, e.g.,enhance stability and/or trimer formation, such as for instance theso-called SOSIP mutations and/or a sequence mutation in the furincleavage site as described above, such as for instance in the ConC_SOSIPsequence shown in SEQ ID NO: 3 and the ConB_SOSIP sequence shown in SEQID NO: 5.

Other non-limiting examples of preferred HIV envelope protein sequencesthat can be used in the invention (as ‘background’ or ‘parent’ molecule,wherein then position 658 is mutated into Val, Ile, Phe, Met, Ala, orLeu) include synthetic HIV Env proteins, for instance comprising theamino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 6 with a mutation ofGlu to Arg at position 166, either of those optionally having furtherSOSIP and/or furin cleavage site mutations as described above. Anothernon-limiting example is SEQ ID NO: 7. Further non-limiting examples aremosaic HIV envelope proteins, such as those having the amino acidsequence of SEQ ID NO: 8 or 9.

In certain embodiments, the parent molecule is a wild-type HIV Envprotein, wherein one or preferably more amino acids have been repairedaccording to methods described herein. Such parent molecules comprise atleast one repair mutation at an amino acid residue that is present atthe corresponding position at a frequency of less than 7.5%, preferablyless than 2%, of HIV Env sequences in a collection of at least 100,preferably at least 500, preferably at least 1000, preferably at least10000, preferably at least 20000, wild-type HIV Env sequences, whereinthe repair mutation is a substitution by an amino acid residue that ispresent at the corresponding position at a frequency of at least 10% ofHIV Env sequences in said collection. Preferably said substitution is byan amino acid residue that is present at the corresponding position at afrequency of at least 15%, at least 20%, at least 25%, of HIV Envsequences in said collection. Preferably, said substitution is by theamino acid residue that is present at the corresponding position mostfrequently in said collection. In certain preferred embodiments, saidparent molecules comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or at least 20 of such repair mutations.Preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or allof the amino acid residues that are present at the correspondingpositions at a frequency of less than 2% of HIV Env sequences in saidcollection are repaired in the parent molecule as compared to thewild-type Env protein, In certain embodiments, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or all of the amino acid residues that arepresent at the corresponding positions at a frequency of less than 7.5%of HIV Env sequences in said collection are repaired in the parentmolecule as compared to the wild-type Env protein. In certainembodiments, the wild-type HIV Env protein is from a clade A, B, or Cstrain, preferably from a clade C strain. As a result of this repairingmutations, the parent molecule will show more resemblance to a HIV Envconsensus sequence than the original wild-type strain, hence therepaired amino acid residue is sometimes referred to herein as‘consensus amino acid’ or ‘consensus residue’. The result of this repairactivity is greatly enhanced properties of the resulting parent moleculewith respect to folding, trimerization, expression, and/or stability,and the resulting molecule is referred to herein as a ‘repaired Envprotein’. The addition of the stabilizing mutations (e.g. (xvi) (Table1), and/or one or more of (i)-(vii) (Table 1), and/or optionally(viii)-(xv) (Table 2)), into such parent molecules leads to an evenfurther improvement in one or more of trimer percentage, trimer yield,stability, broadly neutralizing antibody binding, folding, and theresulting molecules that are derived from wild-type HIV Env proteins arereferred to herein as ‘repaired and stabilized Env protein’. It will beclear to the skilled person that introduction of the stabilizingmutations actually diverts the resulting sequence a bit from a consensussequence, so the net result of greatly enhanced properties of repairedand stabilized HIV Env molecules is based on two entirely differentconcepts.

Mutations resulting in the amino acid at position 658 being replacedwith amino acid Val, Ile, Phe, Met, Ala, or Leu, optionally further withthe indicated amino acids at positions (i)-(vii) described in Table 1can also be used in HIV Env proteins wherein no SOSIP mutations arepresent (e.g. in Env consensus sequences or in Env proteins fromwild-type HIV isolates) and are likely to also improve the trimerizationthereof, as the mutations of the invention are independent from theSOSIP mutations, and mutations described herein in addition were shownto work in several different HIV Env protein backbones. Indeed, it isshown that mutations (i)-(vii) can work in the absence of theSOS-mutations as well as in the absence of the IP-mutation to improveHIV Env trimerization properties.

A recombinant HIV envelope protein according to embodiments of theinvention comprises an HIV envelope protein having certain amino acidresidue(s) at specified positions in the amino acid sequence of an HIVenvelope protein. In particular, it was shown that position 658 in theEnv protein could be mutated to improve trimer formation of the Envprotein, wherein the numbering of the positions is according to thenumbering in gp160 of HIV-1 isolate HXB2. In addition in optionalembodiments, seven positions in the envelope protein were identified, aswell as the particular amino acid residues to be desirable at each ofthe identified positions. Those identified positions in the envelopeprotein sequence include (i) position 651, (ii) position 655, (iii)position 535, (iv) position 589, (v) position 573, (vi) position 204,and (vii) position 647, wherein the numbering of the positions isaccording to the numbering in gp160 of HIV-1 isolate HXB2. An HIV Envprotein according to the invention has Val, Ile, Phe, Met, Ala, or Leu,preferably Val or Ile, more preferably Val, at position 658, andoptionally has the specified amino acid residue(s) in at least one ofthe indicated positions (i)-(vii), preferably at at least two of theindicated positions (i)-(vii), more preferably at at least three of theindicated positions (i)-(vii). The particular amino acid residues thatare desirable to be at each of the identified positions (i)-(vii) areshown in Table 1. The preferred positions of these options are (i),(ii), (iii), (iv), (vi), and/or (vii). Particularly preferred positionsof these options are (i), (ii), (iii), (iv), and/or (vii).

TABLE 1 Desirable Amino Acids at Indicated Positions in the RecombinantHIV Env Proteins According to Certain Embodiments No. Position¹Desirable Amino Acid Residue (i) 651 Phe, Leu, Met, or Trp (preferablyPhe) (ii) 655 Phe, Ile, Met, or Trp (preferably Ile) (iii) 535 Asn orGln (preferably Asn) (iv) 589 Val, Ile, or Ala (preferably Val or Ile,most preferably Val) (v) 573 Phe or Trp (preferably Phe) (vi) 204 Ile(vii) 647 Phe, Met, or Ile (preferably Phe) (xvi) 658 Val, Ile, Phe,Met, Ala, or Leu (preferably Val or Ile, most preferably Val) ¹Accordingto the numbering in gp160 of HIV-1 isolate HXB2

The amino acid sequence of the HIV envelope protein into which the Val,Ile, Phe, Met, Ala, or Leu, at position 658, and optionally the one ormore desirable amino acid (or indicated amino acid) substitutions at theone or more other indicated positions are introduced, is referred to asthe “backbone HIV envelope sequence” or “parent HIV envelope sequence.”For example, if position 658 in the ConC_SOSIP sequence of SEQ ID NO: 3is mutated to Val, then the ConC_SOSIP sequence is considered to be the“backbone” or “parent” sequence. Any HIV envelope protein can be used asthe “backbone” or “parent” sequence into which a novel stabilizingmutation according to an embodiment of the invention can be introduced,either alone or in combination with other mutations, such as theso-called SOSIP mutations and/or mutations in the furin cleavage site.Non-limiting examples of HIV Env protein that could be used as backboneinclude HIV Env protein from a natural HIV isolate, a synthetic HIV Envprotein, or a consensus HIV Env protein, and in certain non-limitingexamples include those comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:9 (wherein for the last four sequences additional amino acids fromnatural Env proteins can be added at the C-term, and position 658 isthen mutated to Val, Ile, Phe, Met, Ala, or Leu; this can be done forany Env sequence that terminates before position 658 according tonumbering in gp160 of HIV-1 isolate HXB2).

According to certain embodiments of the invention, in addition to havingVal, Ile, Phe, Met, Ala, or Leu at position 658, the HIV envelopeprotein can optionally have the indicated amino acid residue at at leastone of the indicated positions selected from the group consisting ofpositions 651, 655, 535, 589, 573, 204, and 647, such as the indicatedamino acid residue of Table 1 at one, two, three, four, five, six, orseven positions. Preferably, the HIV envelope protein is substituted atone, two or three of the indicated positions, and more preferably theHIV envelope protein is substituted at at least two of the indicatedpositions. Even more preferably, the HIV Env protein is substituted atthree of the indicated positions, four of the indicated positions, fiveof the indicated positions, six of the indicated positions, or all sevenof the indicated positions. Preferably, the HIV envelope proteincontains the indicated amino acid residues at at least two of theindicated positions. More preferably, the HIV envelope protein containsthe indicated amino acid residues at three of the indicated positions.In other preferred embodiments, the HIV envelope protein contains theindicated amino acid residues at four, five, six, or all seven of theindicated positions.

Embodiments of HIV Env proteins having the indicated amino acids atmultiple positions are (positions numbered according to numbering ingp160 of HIV-1 isolate HXB2 followed by one letter amino acid code forthe residue present on that position, positions within one HIV Envprotein embodiment divided by commas [e.g. an embodiment of an Envprotein having Ile at position 655 and Val at position 658 is describedas 655I, 658V], while different embodiments (i.e. different HIV Envproteins) are divided by semicolons) include but are not limited to thefollowing.

For Env proteins with the indicated amino acids at two positions: 651F,658V; 651F, 658I; 651F, 658F; 651F, 658M; 651F, 658A; 651F, 658L; 655I,658V; 655I, 658I; 655I, 658F; 655I, 658M; 655I, 658A; 655I, 658L; 655F,658V; 655F, 658I; 655F, 658F; 655F, 658M; 655F, 658A; 655F, 658L; 535N,658V; 535N, 658I; 535N, 658F; 535N, 658M; 535N, 658A; 535N, 658L; 589V,658V; 589V, 658I; 589V, 658F; 589V, 658M; 589V, 658A; 589V, 658L; 589I,658V; 589I, 658I; 589I, 658F; 589I, 658M; 589I, 658A; 589I, 658L; 573F,658V; 573F, 658I; 573F, 658F; 573F, 658M; 573F, 658A; 573F, 658L; 204I,658V; 204I, 658I; 204I, 658F; 204I, 658M; 204I, 658A; 204I, 658L; 647F,658V; 647F, 658I; 647F, 658F; 647F, 658M; 647F, 658A; 647F, 658L. Eachof those embodiments can be present in any HIV Env sequence, such as awild-type isolate, or a SOSIP mutant HIV Env protein, or a consensus HIVEnv protein, or a synthetic HIV Env protein. Each of those embodimentscan be combined according to the invention with one of the preferredamino acids at a second position of one of the other indicated positionsfrom (i)-(vii). Such embodiments, having preferred amino acid residuesat two positions of the indicated positions (i)-(vii) can be combinedwith one of the preferred amino acids at a third position of one of theother indicated positions from (i)-(vii). Such embodiments, havingpreferred amino acid residues at three positions of the indicatedpositions (i)-(vii) can be combined with one of the preferred aminoacids at a fourth position of one of the other indicated positions from(i)-(vii). Such embodiments, having preferred amino acid residues atfour positions of the indicated positions (i)-(vii) can be combined withone of the preferred amino acids at a fifth position of one of the otherindicated positions from (i)-(vii). Such embodiments, having preferredamino acid residues at five positions of the indicated positions(i)-(vii) can be combined with one of the preferred amino acids at asixth position of one of the other indicated positions from (i)-(vii).Such embodiments, having preferred amino acid residues at six positionsof the indicated positions (i)-(vii) can be combined with one of thepreferred amino acids at a seventh position of one of the otherindicated positions from (i)-(vii), such that the Env protein has apreferred amino acid at all seven positions (i)-(vii). Any of thesefurther embodiments having preferred amino acids at two, three, four,five, six or seven of the positions (v)-(vii), can be present in any HIVEnv protein, such as from a wild-type isolate, a SOSIP variant, aconsensus HIV Env protein, a synthetic HIV Env protein, and the like.

Some non-limiting examples of HIV Env proteins with the indicated aminoacids at two of positions (i)-(vii) are: 658V, 651F, 655I; 658I, 651F,655I; 658V, 651F, 535N; 658I, 651F, 535N; 658V, 651F, 589V; 658I, 651F,589V; 658V, 651F, 204I; 658I, 651F, 204I; 658V, 651F, 647F; 658I, 651F,647F; 658V, 655I, 535N; 658I, 655I, 535N; 658V, 655I, 589V; 658I, 655I,589V; 658V, 655I, 204I; 658I, 655I, 204I; 658V, 655I, 647F; 658V, 655I,647F; 658V, 535N, 589V; 658I, 535N, 589V; 658V, 535N, 204I; 658I, 535N,204I; 658V, 535N, 647F; 658I, 535N, 647F; 658V, 589V, 204I; 658I, 589V,204I; 658V, 589V, 647F; 658I, 589V, 647F; 658V, 204I, 647F; 658I, 204I,647F.

Some examples of particularly preferred Env proteins having preferredamino acids at at least two of positions (i)-(vii) include: 658V, 651F,655I; 658I, 651F, 655I; 658V, 651F, 535N; 658I, 651F, 535N; 658V, 651F,589V; 658I, 651F, 589V; 658V, 655I, 535N; 658I, 655I, 535N; 658V, 655I,589V; 658I, 655I, 589V.

Some examples of preferred Env proteins having preferred amino acids atat least three of positions (i)-(vii) include: 658V, 651F, 655I, 535N;658V, 655I, 589V, 535N; 658V, 655I, 573F, 589V; 658V, 655I, 204I, 589V;658V, 651F, 655I, 647F.

Some examples of preferred HIV Env proteins having preferred amino acidresidues at at least four of positions (i)-(vii) include: 651F, 655I,647F, I535N; 651F, 655I, 573F, 589V. A preferred example of an HIV Envprotein comprising the indicated amino acid residues at at least four ofpositions (i)-(vii) comprises 535N, 589V, 651F, 655I. Non-limitingexamples of such HIV Env proteins are provided in SEQ ID NOs: 20, 22,24, 26, 27, 28, 29, 30, 31, and 32. Preferably such HIV Env protein is aclade C HIV Env protein or a clade A HIV Env protein, most preferably aclade C HIV Env protein. In certain embodiments, said HIV Env proteinfurther comprises 588E, i.e. it comprises at least 535N, 588E, 589V,651F, 655I. Non-limiting examples of such HIV Env protein are providedin SEQ ID NOs: 20, 24, 26, 27, 28, 29, 30, 31, and 32. In certainembodiments, said HIV Env further comprises 556P, i.e. it comprises atleast 535N, 556P, 589V, 651F, 655I or at least 535N, 556P, 588E, 589V,651F, 655I. Non-limiting examples of such HIV Env protein are providedin SEQ ID NOs: 22, 24, 26, 27, 29, 30, 31 and 32. Each of theseexemplary embodiments molecules can be further modified to result in anembodiment of the present invention by mutation of the amino acid atposition 658 into V, I, F, M, A, or L, preferably V (except SEQ ID Nos:29, 30 and 31, where the amino acid at position 658 already is V).

In one embodiment, a recombinant HIV Env protein according to theinvention comprises the amino acid sequence of an HIV Env protein havingVal, Ile, Phe, Met, Ala, or Leu, preferably Val or Ile, preferably Val,at position 658, and the indicated amino acid residue at at least one ofthe indicated positions selected from the group consisting of:

-   -   (i) Phe, Leu, Met, or Trp, preferably Phe, at position 651;    -   (ii) Phe, Ile, Met, or Trp, preferably Ile, at position 655;    -   (iii) Asn or Gln, preferably Asn, at position 535;    -   (iv) Val, Ile, or Ala, preferably Val, at position 589;    -   (v) Phe or Trp, preferably Phe, at position 573;    -   (vi) Ile at position 204; and    -   (vii) Phe, Met, or Ile, preferably Phe, at position 647.

For example, the recombinant HIV Env protein can have Val, Ile, Phe,Met, Ala, or Leu, at position 658 and one of Phe, Leu, Met or Trp atposition 651, and optionally, additional indicated amino acid residuesat the additional indicated positions. Preferably, Val, Ile, Phe, Met,Ala, or Leu at position 658, or at least one of the amino acids in(i)-(vii) is introduced into the recombinant HIV Env protein by aminoacid substitution. For example, the recombinant HIV Env protein can beproduced from an HIV Env protein that does not contain Val, Ile, Phe,Met, Ala, or Leu at position 658 or that contains none or only one ofthe amino acid residues in (i)-(vii) above such that all or one or moreof the indicated amino acid residues are introduced into the recombinantHIV Env protein by amino acid substitution.

In certain embodiments, the recombinant HIV Env protein of the inventionfurther comprises (viii) Gln, Glu, Ile, Met, Val, Trp, or Phe atposition 588, wherein Gln or Glu are preferred.

The amino acid sequence of the HIV Env protein into which the abovedescribed substitutions are introduced can be any HIV Env protein knownin the art in view of the present disclosure, such as, for instance anaturally occurring sequence from HIV clade A, clade B, clade C, etc.; amosaic sequence; a consensus sequence, e.g., clade B or clade Cconsensus sequence; a synthetic sequence; or any derivative or fragmentthereof. In certain embodiments of the invention, the amino acidsequence of the HIV Env protein comprises additional mutations, such as,for instance, the so-called SOSIP mutations, and/or a mutation in thefurin cleavage site.

In one particular embodiment, the HIV Env backbone protein is a SOSIPmutant HIV Env protein comprising at least one mutation selected fromthe group consisting of Cys at positions 501 and 605; Pro at position559. In a preferred embodiment, the SOSIP mutant HIV Env proteincomprises Cys at positions 501 and 605, and Pro at position 559.According to this embodiment, a recombinant HIV Env protein comprisesthe amino acid sequence of the SOSIP mutant HIV Env protein and an aminoacid substitution at position 658 resulting in Val, Ile, Phe, Met, Ala,or Leu, preferably Val or Ile, most preferably Val, at this position,and optionally one or more further amino acid substitutions by theindicated amino acid residue at at least one of the indicated positionsselected from the group consisting of:

-   -   (i) Phe, Leu, Met, or Trp, preferably Phe, at position 651;    -   (ii) Phe, Ile, Met, or Trp, preferably Ile, at position 655;    -   (iii) Asn or Gln, preferably Asn, at position 535;    -   (iv) Val, Ile, or Ala, preferably Val, at position 589;    -   (v) Phe or Trp, preferably Phe, at position 573;    -   (vi) Ile at position 204; and    -   (vii) Phe, Met, or Ile, preferably Phe, at position 647.        The SOSIP mutant HIV Env protein can further comprise a mutation        in the furin cleavage site, such as a replacement at positions        608-511 by SEQ ID NO: 10.

In certain embodiments, a recombinant HIV Env protein according to theinvention comprises the amino acid sequence of an HIV Env protein and anamino acid substitution at position 658 resulting in Val, Ile, Phe, Met,Ala, or Leu, preferably Val or Ile, most preferably Val, at thisposition, and optionally one or more further amino acid substitutions bythe indicated amino acid residue at at least one of the indicatedpositions selected from the group consisting of:

-   -   (i) Phe, Leu, Met, or Trp, preferably Phe, at position 651;    -   (ii) Phe, Ile, Met, or Trp, preferably Ile, at position 655;    -   (iii) Asn or Gln, preferably Asn, at position 535;    -   (iv) Val, Ile, or Ala, preferably Val, at position 589;    -   (v) Phe or Trp, preferably Phe, at position 573;    -   (vi) Ile at position 204; and    -   (vii) Phe, Met, or Ile, preferably Phe, at position 647,        wherein the HIV Env protein is selected from the group        consisting of:    -   (1) an HIV Env consensus sequence, such as a clade C or clade B        consensus sequence, e.g. comprising the amino acid sequence of        SEQ ID NO: 2, 3, 4 or 5;    -   (2) a synthetic HIV Env protein.        Preferably, the recombinant HIV Env protein comprises the amino        acid sequence of an HIV Env protein and an amino acid        substitution by the indicated amino acid residue at at least two        of the indicated positions selected from the group consisting of        (i)-(vii) above, such as two positions or three positions.        However, the recombinant HIV Env protein can comprise an amino        acid substitution by the indicated amino acid residue at one or        more of the indicated positions (i)-(vii), such as one, two,        three, four, five, six, or seven of the indicated positions.

In one particular embodiment, the HIV Env backbone protein is an HIV Envconsensus clade C comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 2. Preferably, the HIV consensus clade C sequence of SEQ IDNO: 2 further comprises the so-called SOSIP mutations, i.e., Cys atpositions 501 and 605, and Pro at position 559, and more preferablyfurther comprises the so-called SOSIP mutations and a mutation in thefurin cleavage site, such as for instance a replacement at positions508-511 by SEQ ID NO: 10. In a particularly preferred embodiment, theHIV Env backbone protein comprises the sequence shown in SEQ ID NO: 3,or a sequence at least 95% identical thereto, wherein preferably aminoacids at positions 501, 559, 605, and 508-511 as replaced by SEQ ID NO:10, are not mutated as compared to SEQ ID NO: 3.

In another particular embodiment, the HIV Env backbone protein is an HIVEnv consensus clade B comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 4. Preferably, the HIV consensus clade B sequence of SEQ IDNO: 4 further comprises the so-called SOSIP mutations, i.e., Cys atpositions 501 and 605, and Pro at position 559, and more preferablyfurther comprises the so-called SOSIP mutations and a mutation in thefurin cleavage site, such as for instance a replacement at positions508-511 by SEQ ID NO: 10. In a particularly preferred embodiment, theHIV Env backbone protein comprises the sequence shown in SEQ ID NO: 5,or a sequence at least 95% identical thereto, wherein preferably aminoacids at positions 501, 559, 605, and 508-511 as replaced by SEQ ID NO:10, are not mutated as compared to SEQ ID NO: 5.

In yet another particular embodiment, the HIV Env backbone protein is asynthetic HIV Env protein, e.g. comprising the amino acid sequence of(a) SEQ ID NO: 6; (b) SEQ ID NO: 6 with a mutation of Glu to Arg atposition 166; (c) SEQ ID NO: 7; or (d) SEQ ID NO: 8 or 9, (a) (b) or (d)optionally having further SOSIP (501C, 605C, 559P) and/or furin cleavagesite mutations (508-511RRRRRR) as described above.

In yet other particular embodiments, the HIV Env backbone protein is aHIV Env protein from a wild-type clade A or clade C HIV virus,optionally comprising mutations to repair the sequence according tomethods described herein.

Exemplary combinations of two positions of (i)-(vii) in the HIV Envprotein that can be simultaneously substituted include residues 535,589; 535, 647; and 589, 655; such as for instance in double mutantsI535N, D589V; I535N, E647F; and D589V, K655I. Other double mutantsinclude K655I, I535N; N651F, K655I; and K655I, I573F. An exemplarycombination of three positions in the HIV Env protein that can besimultaneously substituted includes 535,589,655, such as for instance intriple mutant I535N, D589V, K655I. Other triple mutants include K655I,D589V, I573F; and K655I, N651F, I535N.

In certain embodiments of the invention, a recombinant HIV Env proteinaccording to the invention (i.e., having V, I, F, M, A, or L at position658, and optionally one or more indicated amino acid at positions(i)-(vii) above) can further comprise an indicated amino acid residue(e.g. via substitution) at one or more additional indicated positionsselected from the group consisting of positions (viii) 588, (ix) 64 or66, (x) 316, (xi) 201/433, (xii) 556 or 558 or 556 and 558, (xiii)548-568, (xiv) 568, 569 and 636, or (xv) 302, 519 or 520, as shown inTable 2 below. Certain of these amino acid substitutions (e.g. (viii))were found by the present inventors to combine very well with(combinations of) mutations (i)-(vii) as described above. Other of theseamino acid substitutions have been previously reported in theliterature. For example, De Taeye et al. (Cell (2015) 163(7), 1702-15)reported an HIV envelope protein having an E64K and T316W doublemutation, and an HIV Env protein having a 66R mutation; and Kwon et al.(Nat. Struct. Mol. Biol. (2015) 22(7) 522-31) reported an HIV envelopeprotein having an I204C, A433C disulfide substitution; and Guenaga etal. (Immunity (2017) 46, 792-803) reported an HIV envelope proteinhaving L568G, T569G or N636G, and N302Y, F519R, L520R triplesubstitution. However, to the best of the knowledge of the inventors,these previously described mutations were not described in combinationwith any of the novel substitutions described herein, i.e. V, I, F, M, Aor L at position 658, or e.g., the substitutions listed in items(i)-(vii) of Table 1. These amino acid mutations in combination with theamino acid substitutions of the invention can further increase trimeryield and/or the percentage of trimer formation. These amino acidsubstitutions can be introduced into any of the recombinant HIV Envproteins described herein in addition to substitution by the indicatedamino acid residue at position 658, and optionally having furthersubstitutions by the indicated amino acid residue at one or more of theindicated positions as described in Table 1.

TABLE 2 Additional Positions of Amino Acid Substitution and Residue ofSubstitution No. Position¹ Indicated Amino Acid Residue (viii) 588 Gln,Glu, Ile, Met, Val, Trp, or Phe (preferably Gln or Glu) (ix) 64 or 66Lys at position 64; or Arg at position 66 (x) 316 Trp (xi) 201 and 433Cys at both positions (xii) 556 or 558 or Pro at either or bothpositions 556 and 558 (xiii) 548-568 Replacement by shorter and lessflexible loop (HR1 loop) having 7-10 amino acids, preferably a loop of 8amino acids, e.g. having a sequence chosen from any one of (SEQ ID NOs:12-17) (xiv) 568, 569, 636 Gly at any one of these positions, or Gly atboth positions 568 and 636, or Gly at both positions 569 and 636 (xv)302, 519, 520 Tyr at position 302, or Arg at position 519, or Arg atposition 520; or Tyr at position 302 and Arg at position 519; or Tyr atposition 302 and Arg at position 520; or Tyr at position 302 and Arg atboth positions 519 and 520 ¹According to the numbering in gp160 of HIV-1isolate HXB2

The substitutions identified at the indicated positions of the presentinvention [V, I, F, M, A. or L at position 658, and optionally any of(i)-(vii), see e.g. Table 1] are not or rarely present in naturalsequences, are not found in combination in previously reported HIV Envprotein sequences, and were not previously suggested to result inimproved trimerization of the HIV Env protein, improved trimer yieldand/or increased trimer stability. The mutations (ix)-(xi) in Table 2(that were previously reported by others) are all in the gp120 region,to which the trimer specific antibody PGT145 binds. These mutations keepthe trimer closed at the apex (which is at the top of the molecule). Thesubstitutions (xii) and (xiii) are all in the HR1 of gp41. Except forposition 204, the mutations of the present invention in Table 1 are allin the gp41 region (at the bottom part of the molecule), but outside theHR1 region.

Clearly, the previously described mutations did not provide anysuggestion for introduction of the mutations of the present invention,let alone the surprising effects thereof on trimer formation with aclosed apex as measured by PGT145 binding. Apart from the pointmutations (viii)-(xii) in Table 2, it is also possible to replace theHR1 loop of the Env protein (amino acid residues 548-568 in a wild-typesequence, with numbering according to gp160 of the HXB2 isolate) by ashorter and less flexible loop having 7-10 amino acids, preferably aloop of 8 amino acids, e.g. having a sequence chosen from any one of(SEQ ID NOs: 12-17), see e.g. Kong et al (Nat Commun. 2016 Jun. 28;7:12040. doi: 10.1038/ncomms12040) that describes such shorter loopsreplacing the HR1 loop. Such an Env variant, further having theindicated amino acid residues at position 658 (V, I, F, M, A, or L), andoptionally at at least one of the indicated positions (i)-(vii), is alsoan embodiment of the invention. Mutations listed in (viii)-(xiii) can incertain embodiments of the invention be added to HIV Env proteins of theinvention, i.e. having Val, Ile, Phe, Met, Ala, or Leu at position 658.In further embodiments these can be combined with mutations into one ormore of the indicated amino acids at positions (i)-(vii). Also,combinations within the groups (viii)-(xiii) can be made, a non-limitingexample being a combination of mutations (in addition to having Val,Ile, Phe, Met, Ala, or Leu at position 658, and optionally further atleast one mutation of (i)-(vii)) at (viii) and (xii) (e.g. 658V, A556P,K588E). Some non-limiting examples of HIV Env proteins with theindicated amino acid at one of positions (viii)-(xii) are: 658V, 588E;658V, 588Q; 658V, 556P; 658V, 558P; 658V, 201C-433C; 658V; 636G; 658V,568G; 658V, 569G; 658V, 519R; 658V, 520R.

Again, any of those embodiments can be in any HIV Env protein, e.g. awild-type isolate, a consensus Env, a synthetic Env protein, a SOSIPmutant Env protein, etc.

Some preferred combinations of amino acids at indicated positionsinclude 655I, 589V, 573F, 651F, 588E, 535N, 204I; 556P, 655I, 535N,573F, 589V, 204I, 588Q; 204I, 535N, 556P, 588E, 589V, 651F, 655I; 535N,556P, 589V, 651F, 655I; and 535N, 556P, 588E, 589V, 651F, 655I, and eachof those can be combined with an amino acid residue chosen from V, I, F,M, A, or L at position 658.

In certain preferred embodiments, the HIV Env protein comprises asequence that is at least 95% identical to, preferably at least 96%,97%, 98%, 99% identical to, preferably 100% identical to, any one of SEQID NOs: 20, 22, 24, 26, 27, 28, 29, 30, 31 and 32. For determination ofthe % identity, preferably the positions (i)-(xv) of Tables 1 and 2, andpreferably also positions 501, 559 and 605 are not taken into account.Preferably the amino acid residues at those positions are the ones inthe sequences of SEQ ID NO: 20, 22, 24, 26, 27, 28, 29, 30, 31 or 32,respectively, except that for SEQ ID NO: 20, 22, 24, 26, 27, 28 and 32,the amino acid at position 658 is mutated (or added) such that is theresulting amino acid at that position is Val, Ile, Phe, Met, Ala, orLeu, preferably Val or Ile, most preferably Val. It was found that thisstrongly increased trimer percentage and trimer yield of the Envprotein, either alone or in combination with mutations chosen from(i)-(vii) of Table 1 and/or (viii)-(xv) of Table 2 described herein.

According to embodiments of the invention, a recombinant HIV Env proteinhas at least one of (a) an improved percentage of trimer formation, and(b) an improved trimer yield, compared to an HIV Env protein not havingVal, Ile, Phe, Met, Ala, or Leu at position 658 while further beingidentical.

As used herein “improved percentage of trimer formation” means that agreater percentage of trimer is formed when the backbone sequence of theHIV envelope protein contains Val, Ile, Phe, Met, Ala, or Leu atposition 658 as compared to the percentage of trimer that is formed whenthe backbone sequence of the HIV envelope sequence contains a Lysresidue at position 658 (the amino acid present in the majority ofnatural clade C variants of HIV-1 Env at this position; Gln is the mostoccurring amino acid at this position if all strains are considered, andpreferably a greater percentage of trimer formation is also formed whenthe backbone sequence of the HIV envelope protein contains Val, Ile,Phe, Met, Ala, or Leu at position 658 as compared to the percentage oftrimer that is formed when the backbone sequence of the HIV envelopesequence contains a Gln residue at position 658). More generally,“improved percentage of trimer formation” means that a greaterpercentage of trimer is formed when the backbone sequence of the HIVenvelope protein contains one or more of the amino acids substitutionsdescribed in Table 1 and/or 2 as compared to the percentage of trimerthat is formed when the backbone sequence of the HIV envelope sequencedoes not contain such amino acid substitutions. As used herein “improvedtrimer yield” means that a greater total amount of the trimer form ofthe envelope protein is obtained when the backbone sequence of the HIVenvelope protein contains Val, Ile, Phe, Met, Ala, or Leu at position658 as compared to the total amount of trimer form of the envelopeprotein that is obtained when the backbone sequence of the HIV envelopesequence contains a Lys residue at position 658. More generally,“improved trimer yield” means that a greater total amount of the trimerform of the envelope protein is obtained when the backbone sequence ofthe HIV envelope protein contains one or more of the amino acidsubstitutions described in Table 1 and/or 2 as compared to the totalamount of trimer form of the envelope protein that is obtained when thebackbone sequence of the HIV envelope sequence does not contain suchamino acid substitutions.

Trimer formation can be measured by an antibody binding assay usingantibodies that bind specifically to the trimer form of the HIV Envprotein. Examples of trimer specific antibodies that can be used todetect the trimer form include, but are not limited to, the monoclonalantibodies (mAbs) PGT145, PGDM1400, PG16, and PGT151. Preferably, thetrimer specific antibody is mAb PGT145. Any antibody binding assay knownin the art in view of the present disclosure can be used to measure thepercentage of trimer formation of a recombinant HIV Env protein of theinvention, such as ELISA, AlphaLISA, etc.

In a particular embodiment, trimer formation is measured by AlphaLISA.AlphaLISA is a bead-based proximity assay in which singlet oxygenmolecules, generated by high energy irradiation of donor beads, aretransferred to acceptor beads that are within a distance ofapproximately 200 nm with respect to the donor beads. The transfer ofsinglet oxygen molecules to the acceptor beads initiates a cascadingseries of chemical reactions resulting in a chemiluminescent signal thatcan then be detected (Eglen et al. Curr. Chem. Genomics, 2008, 25(1):2-10). For example, recombinant HIV envelope proteins labeled with aFlag-His tag can be incubated with a trimer specific mAb, donor beadsconjugated to the antibody that binds to the trimer specific mAb,nickel-conjugated donor beads, acceptor beads conjugated to an anti-Hisantibody, and acceptor beads conjugated to an anti-Flag antibody. Theamount of trimer formed can be determined by measuring thechemiluminescent signal generated from the pair of donor beadsconjugated to the antibody that binds to the trimer specific mAb and theacceptor beads conjugated to the anti-His antibody. The total amount ofHIV envelope protein expressed can be determined by measuring thechemiluminescent signal generated from the pair of nickel-conjugateddonor beads and anti-Flag-conjugated acceptor beads. For example, theamount of trimer and the total envelope protein expressed can bemeasured by an AlphaLISA assay as described in detail in Example 3. Thepercentage of trimer formation can be calculated by dividing the amountof trimer formed by the total amount of expressed envelope protein.

The amount of trimer formed and the total amount of envelope proteinexpressed can also be determined using chromatographic techniques thatare capable of separating the trimer form from other forms of the HIVenvelope protein, e.g., the monomer form. Examples of such techniquesthat can be used include, but are not limited to size exclusionchromatography multi-angle light scattering (SEC-MALS). According tocertain embodiments, the percentage of trimer formation is determinedusing SEC-MALS. According to certain embodiments, the trimer yield isdetermined using SEC-MALS.

The invention in certain embodiments also provides a method forimproving the trimer formation of an HIV Env protein, the methodcomprising substituting the residue at position 658 (typically Lys) of aparent HIV Env protein with Val, Ile, Phe, Met, Ala, or Leu, preferablyVal or Ile, most preferably Val. This can for instance be done usingstandard molecular biology technology.

Nucleic Acid, Vectors, and Cells

In another general aspect, the invention provides a nucleic acidmolecule encoding a recombinant HIV Env protein according to theinvention, and a vector comprising the nucleic acid molecule. Thenucleic acid molecules of the invention can be in the form of RNA or inthe form of DNA obtained by cloning or produced synthetically. The DNAcan be double-stranded or single-stranded. The DNA can for examplecomprise cDNA, genomic DNA, or combinations thereof. The nucleic acidmolecules and vectors can be used for recombinant protein production,expression of the protein in a host cell, or the production of viralparticles.

According to embodiments of the invention, the nucleic acid encoding therecombinant HIV envelope protein is operably linked to a promoter,meaning that the nucleic acid is under the control of a promoter. Thepromoter can be a homologous promoter (i.e., derived from the samegenetic source as the vector) or a heterologous promoter (i.e., derivedfrom a different vector or genetic source). Examples of suitablepromoters include the human cytomegalovirus immediate early (hCMV IE, orshortly “CMV”) promoter and the Rous Sarcoma virus (RSV) promoter.Preferably, the promoter is located upstream of the nucleic acid withinan expression cassette.

According to embodiments of the invention, a vector can be an expressionvector. Expression vectors include, but are not limited to, vectors forrecombinant protein expression and vectors for delivery of nucleic acidinto a subject for expression in a tissue of the subject, such as aviral vector. Examples of viral vectors suitable for use with theinvention include, but are not limited to adenoviral vectors,adeno-associated virus vectors, pox virus vectors, Modified VacciniaAnkara (MVA) vectors, enteric virus vectors, Venezuelan EquineEncephalitis virus vectors, Semliki Forest Virus vectors, Tobacco MosaicVirus vectors, lentiviral vectors, etc. The vector can also be anon-viral vector. Examples of non-viral vectors include, but are notlimited to plasmids, bacterial artificial chromosomes, yeast artificialchromosomes, bacteriophages, etc.

In certain embodiments of the invention, the vector is an adenovirusvector, e.g., a recombinant adenovirus vector. A recombinant adenovirusvector may for instance be derived from a human adenovirus (HAdV, orAdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus(ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd). Preferably, anadenovirus vector is a recombinant human adenovirus vector, for instancea recombinant human adenovirus serotype 26, or any one of recombinanthuman adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, anadenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53.

The preparation of recombinant adenoviral vectors is well known in theart. For example, preparation of recombinant adenovirus 26 vectors isdescribed, in, e.g., WO 2007/104792 and in Abbink et al., (2007) Virol.81(9): 4654-63. Exemplary genome sequences of adenovirus 26 are found inGenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792.Exemplary genome sequences for rhAd51, rhAd52 and rhAd53 are provided inUS 2015/0291935.

According to embodiments of the invention, any of the recombinant HIVEnv proteins described herein can be expressed and/or encoded by any ofthe vectors described herein. In view of the degeneracy of the geneticcode, the skilled person is well aware that several nucleic acidsequences can be designed that encode the same protein, according tomethods entirely routine in the art. The nucleic acid encoding therecombinant HIV Env protein of the invention can optionally becodon-optimized to ensure proper expression in the host cell (e.g.,bacterial or mammalian cells). Codon-optimization is a technology widelyapplied in the art.

The invention also provides cells, preferably isolated cells, comprisingany of the nucleic acid molecules and vectors described herein. Thecells can for instance be used for recombinant protein production, orfor the production of viral particles.

Embodiments of the invention thus also relate to a method of making arecombinant HIV Env protein. The method comprises transfecting a hostcell with an expression vector comprising nucleic acid encoding arecombinant HIV Env protein according to an embodiment of the inventionoperably linked to a promoter, growing the transfected cell underconditions suitable for expression of the recombinant HIV Env protein,and optionally purifying or isolating the recombinant HIV Env proteinexpressed in the cell. The recombinant HIV Env protein can be isolatedor collected from the cell by any method known in the art includingaffinity chromatography, size exclusion chromatography, etc. Techniquesused for recombinant protein expression will be well known to one ofordinary skill in the art in view of the present disclosure. Theexpressed recombinant HIV Env protein can also be studied withoutpurifying or isolating the expressed protein, e.g., by analyzing thesupernatant of cells transfected with an expression vector encoding therecombinant HIV Env protein and grown under conditions suitable forexpression of the HIV Env protein.

In a preferred embodiment, the expressed recombinant HIV Env protein ispurified under conditions that permit association of the protein so asto form the stabilized trimeric complex. For example, mammalian cellstransfected with an expression vector encoding the recombinant HIV Envprotein operably linked to a promoter (e.g. CMV promoter) can becultured at 33-39° C., e.g. 37° C., and 2-12% CO₂, e.g. 8% CO₂.Expression can also be performed in alternative expression systems suchas insect cells or yeast cells, all conventional in the art. Theexpressed HIV Env protein can then be isolated from the cell culture forinstance by lectin affinity chromatography, which binds glycoproteins.The HIV Env protein bound to the column can be eluted withmannopyranoside. The HIV Env protein eluted from the column can besubjected to further purification steps, such as size exclusionchromatography, as needed, to remove any residual contaminants, e.g.,cellular contaminants, but also Env aggregates, gp140 monomers and gp120monomers. Alternative purification methods, non-limiting examplesincluding antibody affinity chromatography, negative selection withnon-bNAbs, anti-tag purification, or other chromatography methods suchas ion exchange chromatography etc, as well as other methods known inthe art, could also be used to isolate the expressed HIV Env protein.

The nucleic acid molecules and expression vectors encoding therecombinant HIV Env proteins of the invention can be made by any methodknown in the art in view of the present disclosure. For example, nucleicacid encoding the recombinant HIV Env protein can be prepared byintroducing at least one of the amino acid substitutions at theindicated positions into the backbone HIV envelope sequence usinggenetic engineering technology and molecular biology techniques, e.g.,site directed mutagenesis, polymerase chain reaction (PCR), etc., whichare well known to those skilled in the art. The nucleic acid moleculecan then be introduced or “cloned” into an expression vector also usingstandard molecular biology techniques. The recombinant HIV envelopeprotein can then be expressed from the expression vector in a host cell,and the expressed protein purified from the cell culture by any methodknown in the art in view of the present disclosure.

Trimeric Complex

In another general aspect, the invention relates to a trimeric complexcomprising a noncovalent oligomer of three of the recombinant HIV Envproteins according to the invention. The trimeric complex can compriseany of the recombinant HIV Env proteins described herein. Preferably thetrimeric complex comprises three identical monomers (or identicalheterodimers if gp140 is cleaved) of the recombinant HIV Env proteinsaccording to the invention. The trimeric complex can be separated fromother forms of the HIV envelope protein, such as the monomer form, orthe trimeric complex can be present together with other forms of the HIVenvelope protein, such as the monomer form.

Compositions and Methods

In another general aspect, the invention relates to a compositioncomprising a recombinant HIV Env protein, trimeric complex, isolatednucleic acid, vector, or host cell, and a pharmaceutically acceptablecarrier. The composition can comprise any of the recombinant HIV Envproteins, trimeric complexes, isolated nucleic acid molecules, vectors,or host cells described herein.

A carrier can include one or more pharmaceutically acceptable excipientssuch as binders, disintegrants, swelling agents, suspending agents,emulsifying agents, wetting agents, lubricants, flavorants, sweeteners,preservatives, dyes, solubilizers and coatings. The precise nature ofthe carrier or other material can depend on the route of administration,e.g., intramuscular, intradermal, subcutaneous, oral, intravenous,cutaneous, intramucosal (e.g., gut), intranasal or intraperitonealroutes. For liquid injectable preparations, for example, suspensions andsolutions, suitable carriers and additives include water, glycols, oils,alcohols, preservatives, coloring agents and the like. For solid oralpreparations, for example, powders, capsules, caplets, gelcaps andtablets, suitable carriers and additives include starches, sugars,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like. For nasal sprays/inhalant mixtures, the aqueoussolution/suspension can comprise water, glycols, oils, emollients,stabilizers, wetting agents, preservatives, aromatics, flavors, and thelike as suitable carriers and additives.

Compositions of the invention can be formulated in any matter suitablefor administration to a subject to facilitate administration and improveefficacy, including, but not limited to, oral (enteral) administrationand parenteral injections. The parenteral injections include intravenousinjection or infusion, subcutaneous injection, intradermal injection,and intramuscular injection. Compositions of the invention can also beformulated for other routes of administration including transmucosal,ocular, rectal, long acting implantation, sublingual administration,under the tongue, from oral mucosa bypassing the portal circulation,inhalation, or intranasal.

Embodiments of the invention also relate to methods of making thecomposition. According to embodiments of the invention, a method ofproducing a composition comprises mixing a recombinant HIV Env protein,trimeric complex, isolated nucleic acid, vector, or host cell of theinvention with one or more pharmaceutically acceptable carriers. One ofordinary skill in the art will be familiar with conventional techniquesused to prepare such compositions.

HIV antigens (e.g., proteins or fragments thereof derived from HIV gag,pol, and/or env gene products) and vectors, such as viral vectors,expressing the HIV antigens have previously been used in immunogeniccompositions and vaccines for vaccinating a subject against an HIVinfection, or for generating an immune response against an HIV infectionin a subject. As used herein, “subject” means any animal, preferably amammal, most preferably a human, to who will be or has been administeredan immunogenic composition according to embodiments of the invention.The term “mammal” as used herein, encompasses any mammal. Examples ofmammals include, but are not limited to, mice, rats, rabbits, guineapigs, monkeys, humans, etc., preferably a human. The recombinant HIV Envproteins of the invention can also be used as antigens to induce animmune response against human immunodeficiency virus (HIV) in a subjectin need thereof. The immune response can be against one or more HIVclades, such as clade A, clade B, clade C, etc. The compositions cancomprise a vector from which the recombinant HIV Env protein isexpressed, or the composition can comprise an isolated recombinant HIVEnv protein according to an embodiment of the invention.

For example, compositions comprising a recombinant HIV protein or atrimeric complex thereof can be administered to a subject in needthereof to induce an immune response against an HIV infection in thesubject. A composition comprising a vector, such as an adenovirusvector, encoding a recombinant HIV Env protein of the invention, whereinthe recombinant HIV Env protein is expressed by the vector, can also beadministered to a subject in need thereof to induce an immune responseagainst an HIV infection in the subject. The methods described hereinalso include administering a composition of the invention in combinationwith one or more additional HIV antigens (e.g., proteins or fragmentsthereof derived from HIV gag, pol, and/or env gene products) that arepreferably expressed from one or more vectors, such as adenovirusvectors or MVA vectors, including methods of priming and boosting animmune response.

In certain embodiments, the HIV Env protein can be displayed on aparticle, such as a liposome, virus-like particle (VLP), nanoparticle,virosome, or exosome, optionally in combination with endogenous and/orexogenous adjuvants. When compared to soluble or monomeric Env proteinon its own, such particles typically display enhanced efficacy ofantigen presentation in vivo.

Examples of VLPs that display HIV Env protein can be prepared e.g. byco-expressing the HIV Env protein with self-assembling viral proteinssuch as HIV Gag core or other retroviral Gag proteins. VLPs resembleviruses, but are non-infectious because they contain no viral geneticmaterial. The expression of viral structural proteins, such as envelopeor capsid, can result in self-assembly of VLPs. VLPs are well known tothe skilled person, and their use in vaccines is for instance describedin (Kushnir et al, 2012).

In certain preferred embodiments, the particle is a liposome. A liposomeis a spherical vesicle having at least one lipid bilayer. The HIV Envtrimer proteins can for instance be non-covalently coupled to suchliposomes by electrostatic interactions, e.g. by adding a His-tag to theC-terminus of the HIV Env trimer and a bivalent chelating atom such asNi²⁺ or Co²⁺ incorporated into the head group of derivatized lipids inthe liposome. In certain non-limiting and exemplary embodiments, theliposome comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),cholesterol, and the Nickel or Cobalt salt of1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiaceticacid)succinyl] (DGS-NTA(Ni²⁺) or DGS-NTA(Co²⁺)) at 60:36:4 molar ratio.In preferred embodiments, the HIV Env trimer proteins are covalentlycoupled to the liposomal surface, e.g. via a maleimide functional groupintegrated in the liposome surface. In certain non-limiting exemplaryembodiments thereof, the liposome comprises DSPC, cholesterol, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N44-(p-maleimidomethyl)cyclohexane-carboxamidellipid in a molar ratio of 54:30:16. The HIV Env protein can be coupledthereto e.g. via an added C-terminal cysteine in the HIV Env protein.The covalently coupled variants are more stable, elicit high antigenspecific IgG titers and epitopes at the antigenically less relevant‘bottom’ of the Env trimer are masked. Methods for preparing HIV Envtrimers coupled to liposomes, as well as their characterization, areknown and have for instance been described in (Bale et al, 2017),incorporated by reference herein. The invention also provides an HIV Envprotein of the invention fused to and/or displayed on a liposome.

In certain embodiments, a HIV Env protein of the invention is fused toself-assembling particles, or displayed on nanoparticles. Antigennanoparticles are assemblies of polypeptides that present multiplecopies of antigens, e.g. the HIV Env protein of the instant invention,which result in multiple binding sites (avidity) and can provideimproved antigen stability and immunogenicity. Preparation and use ofself-assembling protein nanoparticles for use in vaccines is well-knownto the skilled person, see e.g. (Zhao et al, 2014), (Lopez-Sagaseta etal, 2016). As non-limiting examples, self-assembling nanoparticles canbe based on ferritin, bacterioferritin, or DPS. DPS nanoparticlesdisplaying proteins on their surface are for instance described inWO2011/082087. Description of trimeric HIV-1 antigens on such particleshas for instance been described in (He et al, 2016). Otherself-assembling protein nanoparticles as well as preparation thereof,are for instance disclosed in WO 2014/124301, and US 2016/0122392,incorporated by reference herein. The invention also provides an HIV Envprotein of the invention fused to and/or displayed on a self-assemblingnanoparticle. The invention also provides compositions comprising VLPs,liposomes, or self-assembling nanoparticles according to the invention.

In certain embodiments, an adjuvant is included in a composition of theinvention or co-administered with a composition of the invention. Use ofadjuvant is optional, and may further enhance immune responses when thecomposition is used for vaccination purposes. Adjuvants suitable forco-administration or inclusion in compositions in accordance with theinvention should preferably be ones that are potentially safe, welltolerated and effective in people. Such adjuvants are well known to theskilled person, and non-limiting examples include QS-21, Detox-PC,MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B,Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN,Betafectin, Aluminium salts such as Aluminium Phosphate (e.g. AdjuPhos)or Aluminium Hydroxide, and MF59.

Also disclosed herein are recombinant HIV envelope proteins comprisingan amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4,which represent the HIV envelope consensus clade C and consensus clade Bsequences, respectively. These consensus sequences have not been foundin any naturally occurring sequences, and are thus believed to be novelHIV envelope proteins. A recombinant HIV envelope protein comprising anamino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 canoptionally further comprise the so-called SOSIP mutations and/or amutation in the furin cleavage site, such as, for instance in thosesequences shown in SEQ ID NO: 3, or SEQ ID NO: 3 further comprising Proat position 558 and/or position 556; and SEQ ID NO: 5, or SEQ ID NO: 5further comprising Pro at position 558 and/or position 556. Whendetermining the % identity for these sequences, the amino acids at themutated furin cleavage site and at positions 501, 605, 559, 556 and 558are preferably not taken into account. It was surprisingly found thatsuch proteins are expressed at high levels and have a high level ofstability and trimer formation. Such HIV Env proteins can in certainembodiments be used as backbone proteins, wherein the mutation of K658into V, I, F, M, A, or L can be made to obtain a molecule of theinvention. Isolated nucleic acid molecules encoding these sequences,vectors comprising these sequences operably linked to a promoter, andcompositions comprising the protein, isolated nucleic acid molecule, orvector are also disclosed.

Embodiments

-   -   Embodiment 1 is a recombinant HIV Env protein, that comprises at        position 658 an amino acid chosen from the group consisting of        Val, Ile, Phe, Met, Ala, and Leu, wherein the numbering of the        positions is according to the numbering in gp160 of HIV-1        isolate HXB2.    -   Embodiment 2 is a recombinant HIV Env protein of embodiment 1,        wherein the amino acid at position 658 is Val.    -   Embodiment 3 is a recombinant HIV Env protein of embodiment 1,        wherein the amino acid at position 658 is Ile.    -   Embodiment 4 is a recombinant HIV Env protein of embodiment 1,        wherein the amino acid at position 658 is Met.    -   Embodiment 5 is a recombinant HIV Env protein of embodiment 1,        wherein the amino acid at position 658 is Phe.    -   Embodiment 6 is a recombinant HIV Env protein of embodiment 1,        wherein the amino acid at position 658 is Ala.    -   Embodiment 7 is a recombinant HIV Env protein of embodiment 1,        wherein the amino acid at position 658 is Leu.    -   Embodiment 8 is a recombinant HIV Env protein of any one of        embodiments 1-7, further comprising one or more of the following        amino acid residues:        -   (i) Phe, Leu, Met, or Trp, preferably Phe, at position 651;        -   (ii) Phe, Ile, Met, or Trp, preferably Ile, at position 655;        -   (iii) Asn or Gln, preferably Asn, at position 535;        -   (iv) Val, Ile or Ala, preferably Val or Ile, at position            589;        -   (v) Phe or Trp, preferably Phe at position 573;        -   (vi) Ile at position 204; and        -   (vii) Phe, Met, or Ile, preferably Phe, at position 647,            wherein the numbering of the positions is according to the            numbering in gp160 of HIV-1 isolate HXB2.    -   Embodiment 9 is a recombinant HIV Env protein of embodiment 8,        comprising one or more of the following amino acid residues:        -   (i) Phe, Leu, Met, or Trp, preferably Phe, at position 651;        -   (ii) Phe, Ile, Met, or Trp, preferably Ile, at position 655;        -   (iii) Asn or Gln, preferably Asn, at position 535;        -   (iv) Val, Ile or Ala, preferably Val or Ile, at position            589;        -   (vi) Ile at position 204; and        -   (vii) Phe, Met, or Ile, preferably Phe, at position 647.    -   Embodiment 10 is a recombinant HIV Env protein of embodiment 9,        comprising Phe, Leu, Met, or Trp at position 651.    -   Embodiment 11 is a recombinant HIV Env protein of embodiment 10,        comprising Phe at position 651.    -   Embodiment 12 is a recombinant HIV Env protein of embodiment 9,        comprising Phe, Ile, Met, or Trp at position 655.    -   Embodiment 13 is a recombinant HIV Env protein of embodiment 12,        comprising Ile at position 655.    -   Embodiment 14 is a recombinant HIV Env protein of embodiment 9,        comprising Asn or Gln at position 535.    -   Embodiment 15 is a recombinant HIV Env protein of embodiment 14,        comprising Asn at position 535.    -   Embodiment 16 is a recombinant HIV Env protein of embodiment 9,        comprising Val, Ile or Ala at position 589.    -   Embodiment 17 is a recombinant HIV Env protein of embodiment 16,        comprising Val at position 589.    -   Embodiment 18 is a recombinant HIV Env protein of embodiment 16,        comprising Ile at position 589.    -   Embodiment 19 is a recombinant HIV Env protein of embodiment 9,        comprising Ile at position 204.    -   Embodiment 20 is a recombinant HIV Env protein of embodiment 9,        comprising Phe, Met, or Ile at position 647.    -   Embodiment 21 is a recombinant HIV Env protein of embodiment 20,        comprising Phe at position 647.    -   Embodiment 22 is a recombinant HIV Env protein of embodiment 9,        comprising at least two of the amino acid residues of (i), (ii),        (iii), (iv), (vi), and (vii).    -   Embodiment 23 is a recombinant HIV Env protein of embodiment 22,        comprising at least three of the amino acid residues of (i),        (ii), (iii), (iv), (vi), and (vii).    -   Embodiment 24 is a recombinant HIV Env protein of embodiment 23,        comprising at least four of the amino acid residues of (i),        (ii), (iii), (iv), (vi), and (vii).    -   Embodiment 25 is a recombinant HIV Env protein of embodiment 24,        comprising at least five of the amino acid residues of (i),        (ii), (iii), (iv), (vi), and (vii).    -   Embodiment 26 is a recombinant HIV Env protein of embodiment 25,        comprising all six of indicated amino acid residues of (i),        (ii), (iii), (iv), (vi), and (vii).    -   Embodiment 27 is a recombinant HIV Env protein of embodiment 9,        comprising Val at position 658 and Ile at position 655.    -   Embodiment 28 is a recombinant HIV Env protein of embodiment 9,        comprising Val at position 658 and Phe at position 651.    -   Embodiment 29 a recombinant HIV Env protein of embodiment 9,        comprising Ile at position 658 and Ile at position 655.    -   Embodiment 30 a recombinant HIV Env protein of embodiment 9,        comprising Ile at position 658 and Phe at position 651.    -   Embodiment 31 a recombinant HIV Env protein of embodiment 9,        comprising Val at position 658 and Phe at position 655.    -   Embodiment 32 is a recombinant HIV Env protein of any one of        embodiments 27, 29, or 31, further comprising Phe at position        651.    -   Embodiment 33 is a recombinant HIV Env protein of any one of        embodiments 1-32, wherein the HIV Env is from a clade C HIV.    -   Embodiment 34 is a recombinant HIV Env protein of any one of        embodiments 1-33, comprising a HIV Env parent molecule that has        been mutated at one or more of the indicated positions to obtain        the indicated amino acid residue at said one or more positions,        wherein the parent molecule has a consensus HIV Env sequence.    -   Embodiment 35 is a recombinant HIV Env protein of any one of        embodiments 1-33, comprising a HIV Env parent molecule that has        been mutated at one or more of the indicated positions to obtain        the indicated amino acid residue at said one or more positions,        wherein the parent molecule is a synthetic Env protein.    -   Embodiment 36 is a recombinant HIV Env protein of any one of        embodiments 1-33, comprising a HIV Env parent molecule that has        been mutated at one or more of the indicated positions to obtain        the indicated amino acid residue at said one or more positions,        wherein the parent molecule is a wild-type HIV Env protein,        preferably of clade C, comprising at least one repair mutation        at an amino acid residue that is present at the corresponding        position at a frequency of less than 7.5%, preferably less than        2%, of HIV Env sequences in a collection of at least 100,        preferably at least 1000, preferably at least 10000, wild-type        HIV Env sequences, wherein the repair mutation is a substitution        by an amino acid residue that is present at the corresponding        position at a frequency of at least 10% of HIV Env sequences in        said collection and preferably the repair mutation is a        substitution by the amino acid residue that is present at the        corresponding position most frequently in said collection.    -   Embodiment 37 is a recombinant HIV Env protein of any one of        embodiments 1-36, further comprising Cys at positions 501 and        605, or Pro at position 559, preferably Cys at positions 501 and        605 and Pro at position 559.    -   Embodiment 38 is a recombinant HIV Env protein of embodiment 36,        comprising Cys at positions 501 and 605 and Pro at position 559.    -   Embodiment 39 is a recombinant HIV Env protein of any one of        embodiments 1-38, further comprising one or more of the        following:        -   (viii) Gln, Glu, Ile, Met, Val, Trp, or Phe, preferably Gln            or Glu, at position 588;        -   (ix) Lys at position 64 or Arg at position 66 or both Lys at            position 64 and Arg at position 66;        -   (x) Trp at position 316;        -   (xi) Cys at both positions 201 and 433;        -   (xii) Pro at position 556 or 558 or at both positions 556            and 558;        -   (xiii) replacement of the loop at amino acid positions            548-568 (HR1-loop) by a loop having 7-10 amino acids,            preferably a loop of 8 amino acids, e.g. having a sequence            chosen from any one of (SEQ ID NOs: 12-17);        -   (xiv) Gly at position 568, or Gly at position 569, or Gly at            position 636, or Gly at both positions 568 and 636, or Gly            at both positions 569 and 636; and/or        -   (xv) Tyr at position 302, or Arg at position 519, or Arg at            position 520, or Tyr at position 302 and Arg at position            519, or Tyr at position 302 and Arg at position 520, or Tyr            at position 302 and Arg at both positions 519 and 520.    -   Embodiment 40 is a recombinant HIV Env protein of embodiment 39,        comprising Pro at position 556.    -   Embodiment 41 is a recombinant HIV Env protein of embodiment 39,        comprising Pro at position 558.    -   Embodiment 42 is a recombinant HIV Env protein of embodiment 39,        comprising Pro at positions 556 and 558.    -   Embodiment 43 is a recombinant HIV Env protein of any one of        embodiments 1-42, further comprising a mutation in a furin        cleavage site of the HIV Env protein.    -   Embodiment 44 is a recombinant HIV Env protein of embodiment 43,        wherein the mutation in a furin cleavage site comprises a        replacement at positions 508-511 by RRRRRR (SEQ ID NO: 10).    -   Embodiment 45 is the recombinant HIV Env protein of any of        embodiments 1-44, being a gp140 or gp160 protein.    -   Embodiment 46 is the recombinant HIV Env protein of embodiment        45, being a gp140 protein.    -   Embodiment 47 is the recombinant HIV Env protein of any of        embodiments 1-46, wherein the recombinant HIV Env protein has at        least one of (a) an improved percentage of trimer formation        and (b) an improved trimer yield, compared to a further        identical HIV Env protein except that it that comprises Lys at        position 658.    -   Embodiment 48 is the recombinant HIV Env protein of embodiment        47, wherein trimer formation is measured by size exclusion        chromatography with multi-angle light scattering (SEC-MALS).    -   Embodiment 49 is the recombinant HIV Env protein of any of        embodiments 1 to 48, comprising, in addition to V, I, F, M, A,        or L at position 658, a combination of amino acids chosen from        the group consisting of:        -   (a) 655I, 589V, 573F, 651F, 588E, 535N, 204I;        -   (b) 556P, 655I, 535N, 573F, 589V, 204I, 588Q;        -   (c) 204I, 535N, 556P, 588E, 589V, 651F, 655I;        -   (d) 535N, 556P, 589V, 651F, 655I; and        -   (e) 535N, 556P, 588E, 589V, 651F, 655I.    -   Embodiment 50 is a recombinant HIV Env protein of any of        embodiments 1 to 49, comprising an amino acid sequence that is        at least 95%, 96%, 97%, 98%, 99% identical to any one of SEQ ID        NOs: 3, 5, 20, 22, 24, 26, 27, 28, 29, 30, 31, or 32, preferably        at least 98% identical to any one of SEQ ID NOs: 20, 22, 24, 26,        27, 28, 29, 30, 31, or 32, or 100% identical to any one of SEQ        ID NOs: 29, 30, or 31.    -   Embodiment 51 is a trimeric complex comprising a noncovalent        oligomer of three of the recombinant HIV Env proteins of any of        embodiments 1-50.    -   Embodiment 52 is a particle, for example a liposome or        nanoparticle, e.g. a self-assembling nanoparticle, displaying        the recombinant HIV Env protein of any one of embodiments 1-50        or the trimeric complex of embodiment 51.    -   Embodiment 53 is an isolated nucleic acid molecule encoding a        recombinant HIV Env protein of any of embodiments 1-50.    -   Embodiment 54 is a vector comprising the isolated nucleic acid        molecule of embodiment 53 operably linked to a promoter.    -   Embodiment 55 is the vector of embodiment 54, which is an        adenovirus vector.    -   Embodiment 56 is a host cell comprising the isolated nucleic        acid molecule of embodiment 53 or the vector of embodiment 54 or        55.    -   Embodiment 57 is a method of producing a recombinant HIV Env        protein, comprising growing the host cell of embodiment 56 under        conditions suitable for production of the recombinant HIV Env        protein.    -   Embodiment 58 is a method of producing a recombinant HIV Env        protein comprising obtaining an expression vector comprising the        isolated nucleic acid of embodiment 53 operably linked to a        promoter; transfecting a cell with the expression vector;        growing the transfected cell under conditions suitable for        expression of the recombinant HIV Env protein; and purifying the        recombinant HIV Env protein under conditions that permit        formation of a stabilized trimeric complex.    -   Embodiment 59 is a method of producing a recombinant HIV Env        protein according to any one of embodiments 1 to 50, comprising        introducing an amino acid substitution into a backbone HIV        envelope protein sequence at position 658 such that the        resulting amino acid at that position is Val, Ile, Phe, Met,        Ala, or Leu, preferably Val or Ile, most preferably Val.    -   Embodiment 60 is the method according to embodiment 59, wherein        a nucleotide sequence encoding the amino acid substitution is        introduced into nucleic acid encoding the backbone HIV envelope        protein sequence.    -   Embodiment 61 is the method of embodiments 59 or 60, wherein the        backbone HIV envelope protein sequence is selected from the        group consisting of;    -   SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; and    -   a wild-type HIV Env protein having mutations that result in at        least (a), (b) or (c), preferably at least two of (a), (b) and        (c), most preferably (a), (b) and (c) of the following:    -   (a) Cys at positions 501 and 506 and Pro at position 559,    -   (b) having SEQ ID NO: 10 replacing amino acids 508-511, and/or    -   (c) at least one repair mutation at an amino acid residue that        is present at the corresponding position at a frequency of less        than 7.5%, preferably less than 2%, of HIV Env sequences in a        collection of at least 100, preferably at least 1000, preferably        at least 10000, wild-type HIV Env sequences, wherein the repair        mutation is a substitution by an amino acid residue that is        present at the corresponding position at a frequency of at least        10% of HIV Env sequences in said collection and preferably the        repair mutation is a substitution by the amino acid residue that        is present at the corresponding position most frequently in said        collection.    -   Embodiment 62 is a composition comprising the recombinant HIV        Env protein of any of embodiments 1-50, the trimeric complex of        embodiment 51, the particle of embodiment 52, the isolated        nucleic acid molecule of embodiment 53, or the vector of        embodiment 54 or 55, and a pharmaceutically acceptable carrier.    -   Embodiment 63 is a composition of embodiment 62, further        comprising an adjuvant.    -   Embodiment 64 is a method of producing the composition of        embodiment 62, comprising mixing the recombinant HIV Env        protein, trimeric complex, particle, isolated nucleic acid, or        vector with one or more pharmaceutically acceptable carriers.    -   Embodiment 65 is a method of vaccinating a subject against an        HIV infection comprising administering to the subject a        composition comprising the recombinant HIV envelope protein of        any one of embodiments 1-50, the trimeric complex of embodiment        51, the particle of embodiment 52, the isolated nucleic acid of        embodiment 53, or the vector of embodiment 54 or 55.    -   Embodiment 66 is a method of producing an immune response        against an HIV infection in a subject in need thereof,        comprising administering to the subject a composition comprising        the recombinant HIV envelope protein of any one of embodiments        1-50, the trimeric complex of embodiment 51, the particle of        embodiment 52, the isolated nucleic acid of embodiment 53, or        the vector of embodiment 54 or 55.    -   Embodiment 67 is the particle of embodiment 52, wherein the        particle is a liposome.    -   Embodiment 68 is the particle of embodiment 52, wherein the        particle is a self-assembling nanoparticle.

EXAMPLES Example 1: Generation of HIV Envelope Clade C and Clade BConsensus Sequence HIV Envelope Clade C Consensus Sequence

An HIV clade C envelope (Env) protein consensus sequence was developedas the backbone sequence for studying the effects of various mutationson trimer formation of the HIV Env proteins. A sequence alignment of3,434 envelope protein sequences from known HIV viral isolates wasdownloaded from the Los Alamos Database(http://www.hiv.lanl.gov/content/index). From the 3,434 sequences, 1,252sequences of clade C only were selected to generate the HIV clade C Envprotein consensus sequence. At positions for which a consensus residuecould not be clearly identified based on the alignment, the consensussequence was used to identify the closest wild-type sequences by a BLASTsearch. The consensus residue at these positions was then selected asthe amino acid in the closest wild-type sequences identified from theBLAST search. The HIV Env clade C consensus sequence is shown in SEQ IDNO: 2.

The HIV Env clade C consensus sequence was further modified byintroducing the so-called SOSIP mutations, which include cysteineresidues at positions 501 and 605 and a proline residue at position 559,as well as optimizing the furin cleavage site by replacing the furinsite at residues 508-511 with 6 arginine residues. Further, Val atposition 295 was mutated into an Asn (V295N), to create an N-linkedglycosylation site present in the majority of HIV strains and that canimprove binding to certain antibodies used in some experiments.Additionally, the C-terminus was truncated at residue 664, resulting ina sequence encoding a soluble HIV gp140 protein. All positions ofsubstitution/modification described above are relative to the numberingin gp160 of HIV-1 isolate HXB2. The resulting HIV gp140 sequence,referred to as “ConC_SOSIP,” is shown in (SEQ ID NO: 3). The ConC_SOSIPsequence was used as the backbone or parent HIV envelope sequence intowhich additional mutations, e.g., single and double amino acidsubstitutions, were introduced to produce recombinant HIV Env proteinsdescribed herein.

HIV Envelope Clade B Consensus Sequence

An HIV Env clade B consensus sequence was generated using a similarprocedure as that described above for generating the HIV Env clade Cconsensus sequence. The clade B consensus sequence was generated using1,708 clade B envelope protein sequences from known clade B viralisolates. The HIV Env clade B consensus sequence is shown in SEQ ID NO:4.

The HIV Env clade B consensus sequence was further modified byintroducing the so-called SOSIP mutations, optimizing the furin cleavagesite by replacing the furin site with 6 arginine residues, andtruncating the C-terminus at residue 664, as described above, resultingin a sequence encoding a soluble HIV gp140 clade B consensus sequence.The resulting HIV gp140 Env protein sequence, referred to as“ConB_SOSIP” is shown in (SEQ ID NO: 5).

It was found that the consensus-based molecules had improved expressionlevels over molecules based on natural isolates, and moreover alreadyhad improved trimerization levels.

Example 2: Expression and Purification of Recombinant HIV Env Protein

Recombinant HIV Env proteins were expressed and purified as solublegp140 proteins. Single mutations (amino acid substitutions) andcombinations thereof (e.g., double and triple mutations) were introducedinto the ConC_SOSIP backbone consensus sequence to generate a series ofrecombinant HIV Env protein variants.

Generation and Expression of HIV Gp140 Env Constructs and Variants

DNA encoding the HIV clade C Env consensus sequence ConC_SOSIP shown inSEQ ID NO: 3 was synthesized and codon-optimized at GenScript(Piscataway, NJ 08854) or Gene Art (Life Technologies, Carlsbad, CA).The codon-optimized sequence was then cloned into the vector pcDNA2004to generate an HIV clade C gp140 Env construct, which was used as thebackbone HIV envelope sequence for introducing further mutations.Mutations were introduced into the ConC_SOSIP backbone sequence by sitedirected mutagenesis and polymerase chain reaction (PCR) performed onthe pcDNA2004 HIV clade C gp140 Env construct. HEK-Expi293F cells orHEK293F cells were transiently transfected with 90% of the pcDNA2004vector encoding the ConC_SOSIP sequence or variant thereof and 10% of apcDNA2004 vector encoding the furin protease (furin-pCDNA2004) accordingto the manufacturer's instructions. The transfected cells were culturedfor 5 days at 37° C. and 10% CO₂. Culture supernatants were spun for 10minutes at 1250×g. The spun supernatant was subsequently sterilefiltered using a 0.22 μm vacuum filter and stored at 4° C. until furtheruse. For expressions in 96-well format the cells were cultured for 3days at 37° C. and 10% CO₂. 4 uL of Optimem (culture medium) was mixedwith 4 uL 100 ng/uL DNA and 8 uL Expi293F mix (54 uL/mL Optimem) asadded and incubated for 20 minutes. Subsequently 200 uL/well Expi293Fcells were added at 2.5×10E6 cells/mL. The culture supernatant washarvested and spun for 5 minutes at 300 g to remove cells and cellulardebris. The spun supernatant was subsequently sterile filtered using a0.22 μm vacuum filter and stored at 4° C. until further use.

Purification of HIV gp140 Env Protein

HIV gp140 Env protein expressed from the pcDNA2004 vector was purifiedaccording to a two-step purification protocol using a Galantusnivalis-lectin column (Vectorlabs, AL-1243) for the initialpurification, and a Superdex200 Increase column (GE) in a subsequentstep to remove residual contaminants. For the initial step using theGalantus nivalis-lectin column, culture supernatant was diluted withbuffer (40 mM Tris, 500 mM NaCl pH 7.5) and passed over a 4 mL CVTricorn 10-50 Lectin Agarose Column at a rate of 4 mL per minute.Subsequently, the column was washed with four column volumes buffer (40mM Tris, 500 mM NaCl pH 7.5) and eluted with four column volumes of 40mM Tris, 500 mM NaCl, and 1 M mannopyranoside pH 7.5 with an upflow of1.6 mL/min, meaning that the direction of flow has been changed fromdown to up to increase the rate of elution of envelope protein anddecrease the elution volume. The eluate was concentrated using a spinconcentrator (50K, Amicon Ultra, Millipore).

The HIV gp140 Env protein was further purified on a Superdex200 columnusing 50 mM Tris, 150 mM NaCl pH 7.4 as running buffer. The second peakthat eluted from the column contained the HIV gp140 Env protein. Thefractions containing this peak were pooled, and the identity of the peakconfirmed as HIV gp140 Env protein using Western blot and SDS-PAGE,and/or SEC-MALS analysis. The concentration of the purified HIV gp140Env protein was determined by measuring the optical density at 280 nm,and the purified HIV gp140 Env protein was stored at 4° C. until furtheruse.

SDS-PAGE and Western Blotting Analysis

Cell culture supernatants containing expressed HIV gp140 Env protein andpurified HIV gp140 Env protein samples were analyzed on 4-12% (w/v)Bis-Tris NuPAGE gels, 1×MOPS (Life Technologies) under reducing ornon-reducing conditions, and blotted using the iBlot technology (LifeTechnologies). All procedures were performed according to themanufacturer's instructions. For purity analysis, the gels were stainedwith Krypton Infrared Protein Stain (Thermo Scientific) or SYPRO Rubiprotein stain (Bio-Rad). For Western blotting analysis, membranes wereprobed with an anti-6×-Histidine-Tag antibody (anti-His-HRP). The gelsand the blot membranes were scanned on an Odyssey instrument (Li-Cor),and images were analyzed using Odyssey 3.0 software (Li-Cor).

Example 3: Screening of Recombinant HIV Gp140 Env Variants for TrimerYield and Percentage of Trimer Formation

The recombinant HIV Env protein variants generated in Example 2 werescreened for trimer formation to identify those mutations that improvedthe percentage of trimer formed and/or improved trimer yields relativeto the ConC_SOSIP backbone sequence. High throughput screening of trimerpercentage and trimer yields was conducted using an AlphaLISA assay toevaluate the binding of a panel of broadly neutralizing HIV antibodies(bNAbs) and non-bNAbs to the recombinant HIV Env proteins. The resultsof the AlphaLISA assay were confirmed by size exclusion chromatographyand multi-angle light scattering (SEC-MALS).

AlphaLISA® Assay Analysis

Total expression of the HIV gp140 Env protein and the total amount ofcorrectly folded native trimer of over 200 HIV gp140 variants withsingle amino acid substitutions introduced into the ConC_SOSIP sequencegenerated as described in Example 2 were measured in cell culturesupernatant by AlphaLISA assay. HIV gp140 variants containing double andtriple mutations were also tested. The HIV Env protein having theConC_SOSIP sequence without any additional mutations was tested forcomparison.

The following monoclonal antibodies (mAbs) were inter alia used foranalysis: mAb PGT145, mAb PGDM1400, mAb PG16, mAb PGT151, mAb 35022, mAbPGT128, mAb PG9, mAb F105, mAb B6, mAb 447-52d, mAb 14e, and mAb 17b.MAbs 447-52D (AB014), PG9 (AB015), and PG16 (AB016) were purchased fromPolymun Scientific Immunbiologische Forschung GmbH (Klosterneuburg,Austria). The non-neutralizing antibody b6 was obtained from Dennis R.Burton (The Scripps Research Institute, La Jolla, CA), and thenon-neutralizing antibody 14e was obtained from James E. Robinson(Tulane University, New Orleans, LA). For mAbs PGT145 (PDB: 3U1S),PGDM1400 (PDB: 4RQQ), PGT151 (PDB: 4NUG), 35022 (PDB: 4TVP), F105 (PDB:1U6A), PGT128 (PDB: 3TYG), and 17b (PDB: 4RQS) nucleic acids encodingthe published sequences were cloned into an expression vector andproduced for evaluation of the HIV Env proteins. With the exception ofmAbs F105, B6, 447-52d, 14e, and 17b, the antibodies used for analysisare broadly neutralizing antibodies (bNAbs). bNAbs are capable ofneutralizing multiple HIV viral strains. Of the bNAbs, PGT145, PGDM1400,and PG16 are apex binders and are trimer specific. PGT151 is also trimerspecific, but binds at the interface of two protomers of gp120 and gp41,and is cleavage dependent. Binding of non-bNAbs is indicative ofincorrect folding or an open trimer conformation.

Protein folding was also tested by measuring the binding of soluble HIVgp140 Env protein variants to an antibody (mAb 17b) known to bind theco-receptor binding site of the HIV envelope protein, which is exposedonly after binding of CD4 (data not shown). In particular, solublereceptor CD4 (sCD4) was used in combination with mAb 17 to evaluateCD4-induced conformational change. Binding of mAb 17b to the HIV gp140Env protein variant without prior CD4 binding to the envelope protein isan indication of partially unfolded or pre-triggered envelope protein(i.e., an unstable Env that adopts the “open” conformation in theabsence of CD4 binding).

For the AlphaLISA assay, HIV gp140 Env constructs in the pcDNA2004vector containing a linker followed by a sortase A tag followed by aFlag-tag followed by a flexible (G45)₇ linker and ending with a His-tag,were prepared (the sequence of the tag, which was placed at theC-terminus of the HIV Env protein, is provided in SEQ ID NO: 19). TheHIV gp140 Env constructs were expressed in HEK-Expi293 cells, which werecultured for three days in 96 well plates (200 μL/well). Crudesupernatants were diluted 120 times in AlphaLISA buffer (PBS+0.05%Tween-20+0.5 mg/mL BSA). For mAb 17b based assays, supernatants werediluted 12 times. Then, 10 μL of each dilution were transferred to a96-well plate and mixed with 40 μL acceptor beads, donor beads, and oneof the above listed mAbs. The donor beads were conjugated to ProtA (Cat#: AS102M, Lot #1831829, Perkin Elmer), which binds to the mAb. Theacceptor beads were conjugated to an anti-His antibody (Cat #: AL128M,Perkin Elmer), which binds to the His-tag of the construct. Forquantification of the total protein yield, including all forms of theenvelope protein, a combination of Nickel-conjugated donor beads (Cat #:AS101M, Perkin Elmer) for detection of the His-tag together withanti-Flag antibody-conjugated acceptor beads (Cat #: AL112R, PerkinElmer) for detection of the Flag tag were used. For the tests using mAb17b in combination with sCD4-His, a combination of ProtA donor beads andanti-Flag acceptor beads were used (data not shown). One sample wasmixed with donor and acceptor beads to detect trimer formation, and asecond sample of the same Env variant was mixed with nickel-conjugateddonor beads and anti-Flag conjugated acceptor bead to measure the totalamount of protein expressed (i.e., total protein yield).

The mixture of the supernatant containing the expressed HIV gp140 Envprotein, the mAb, donor beads, and acceptor beads was incubated at roomtemperature for 2 hours without shaking. Subsequently, thechemiluminescent signal was measured with a Synergy NEO plate readerinstrument (BioTek). The average background signal attributed to mocktransfected cells was subtracted from the AlphaLISA counts measured foreach the HIV gp140 Env variants. Then, the whole data set was divided bysignal measured for the HIV Env protein having the ConC_SOSIP backbonesequence signal to normalize the signal for each of the HIV gp140 Envvariants tested to the backbone. Binding data for each of the HIV gp140Env variants to the trimer specific mAb PGT145 was used to determine thepercentage of trimer formation and trimer yield for each of thevariants. Binding to the other mAbs was used to evaluate the generalbinding pattern of the HIV Env variants to bNAbs and non-bNAbs (notshown).

The percentage of trimer formation for each of the HIV Env variants wascalculated by dividing the normalized chemiluminescent signal obtainedfrom sample mixture of HIV Env variant, the mAb PGT145, ProtA-conjugateddonor beads, and anti-His-conjugated acceptor beads, by the normalizedchemiluminescent signal obtained from the sample mixture of the HIV Envvariant, anti-His-conjugated donor beads and anti-Flag-conjugatedacceptor beads.

Trimer yield for each of the HIV Env variants was determined relative tothe trimer yield for the HIV Env protein having ConC_SOSIP backbonesequence without any additional mutations. The normalizedchemiluminescent signal obtained from the binding of mAb PGT145 to theConC_SOSIP envelope protein was set to 1, and the normalizedchemiluminescent signal obtained from the binding of mAb PGT145 to eachof the HIV gp140 proteins was normalized to this value.

Results of AlphaLISA Assay Analysis-Trimer Percentage and Trimer Yields

The percentage of trimer formation as determined by the AlphaLISA assayfor several single, double, and triple amino acid substitutions from thelist of (i)-(vii) in Table 1 above in the ConC_SOSIP backbone sequenceis shown in FIG. 2A. Of the about 200 HIV gp140 Env variants containingsingle amino acid substitutions that were tested, seven positions ofsubstitution were identified for which the percentage of trimer formedincreased by at least 25% relative to the percentage of trimer formedfor the ConC_SOSIP backbone sequence without any additional amino acidsubstitutions.

The results shown in FIG. 2A demonstrate that the seven preferredpositions of substitution for which a significant increase in thepercentage of trimer formation was observed include N651, K655, 1535,D589, 1573, A204, and E647 according to the numbering in gp160 of HIV-1isolate HXB2. In particular, the single amino acid substitutions thatresulted in the most improved percentage of trimer formation includedN651F, K655I(/F/W) (although there was also one experiment in whichK655F did not appear to result in improvement), I535N, D589V(/A), I573F,A204I, E647F. Some mutations that were tested in combination withseveral of these mutations, included K588Q/E, I556P and A558P, and thesefurther improved the trimer percentage of mutants with preferred aminoacids at positions (i)-(vii) of Table 1 in this experiment.

All double substitutions tested in this experiment had a higherpercentage of trimer formation than the corresponding singlesubstitutions, and all triple substitutions tested had a higherpercentage of trimer formation than the corresponding single and doublemutations (FIG. 2A). These unpredictable and surprising results indicatethat these mutations could display a form of synergy in theseexperiments with respect to trimerization of the envelope protein.

In addition to improved percentage of trimer formation, an increasedtrimer yield is also desirable. Therefore, the trimer yield of HIV gp140variants containing single, double, and triple mutations in theConC_SOSIP backbone sequence was also determined by the AlphaLISA assay.The results are shown in FIG. 2B. Most HIV gp140 variants containingsingle mutations (exceptions were I535N, D589A and D589I), had a highertrimer yield than the ConC_SOSIP envelope protein. However, the moreaccurate SEC-MALS analysis of the I535N mutant, as described below,showed an increase in trimer yield. Moreover, additional mutations incombination with I535N, such as D589V, resulted in the same trimer yieldobserved for the envelope protein having that particular additionalsubstitution in the absence of the I535N mutation. The trimer yield ofthe variants with double mutations was also increased where each of thesingle mutation variants had a higher trimer yield than the ConC_SOSIPenvelope protein (FIG. 2B).

The percentage of trimer formation for HIV gp140 variants with doublemutations in the ConC_SOSIP backbone that were previously described inthe literature was also tested, including the E64K, T316W doublesubstitution described by (De Taeye et al., supra), and the disulfidedouble substitution I204C, A433C described by (Kwon et al., supra). TheE64K, T316W double substitution resulted in a lower percentage of trimerformation than the ConC_SOSIP envelope protein, i.e., 15% (data notshown). Although the disulfide double substitution I204C, A433Cincreased the trimer percentage to 43% (data not shown), doublesubstitutions described herein, such as I535N/K588E, K588Q/D589V,K655I/K588E, I535N/D589V, I535N/E647F, D589V/K655I, and 1535N/K655I(FIG. 2A) resulted in an even greater percentage of trimer formation inthe AlphaLISA experiment.

Additional mutations (proline at residues 558 and/or 556) were alsointroduced into the ConC_SOSIP backbone, and the percentage of trimerformation and trimer yield measured for these HIV gp140 Env proteins.Both the single substitutions of Pro at position 558 or 556, and thedouble substitution of proline at both positions 556 and 558 in additionto the SOSIP mutations already contained in the ConC_SOSIP backbone(i.e., Cys at positions 501 and 605, and Pro at position 559) increasedthe percentage of trimer formation and trimer yield (data not shown).Indeed, introduction of one or more of the novel amino acid stabilizingsubstitutions of the invention in the ConC_SOSIP backbone furthercomprising Pro residues at positions 558 and/or 556 further improves thepercentage of trimer formation and/or trimer yield (e.g. FIG. 2A, e.g.A558P/I535N, K655I/L556P, and several triple mutants including the A558Pmutation).

Binding data of the HIV gp140 Env variants to the other bNAbs andnon-bNAbs demonstrated that most of the single, double and triplemutations tested which increased trimer yield and the percentage oftrimer formation, such as those listed in FIGS. 2A and 2B, also hadincreased binding to bNAbs, and the same or decreased binding tonon-bNAbs relative to the amount of binding observed to the bNAbs andnon-bNAbs for the HIV envelope protein having the ConC_SOSIP backbonesequence (data not shown). For vaccine development, increased binding tobNAbs and reduced binding to non-bNAbs is preferred. The data thusdemonstrates that the HIV envelope proteins comprising the amino acidsubstitutions at positions (i)-(vii) indicated in Table 1 above havedesirable properties with respect to binding patterns to broadlyneutralizing and non-broadly neutralizing antibodies.

SEC-MALS Analysis

SEC-MALS analysis was also used to verify the trimer yield andpercentage of trimer formation for the HIV gp140 variants screened usingthe AlphaLISA assay. The HIV gp140 variants were expressed in 30 mLscale cultures and purified by applying the cell free supernatants on200 μl Galanthus nivalis lectin beads (Vectorlab Cat #AL-1243) inPolyprep gravity flow columns (Biorad Cat #731-1550). The beads werewashed with 2 ml binding buffer (40 mM Tris, 500 mM NaCl pH 7.4). Theproteins were eluted using 250-500 μl of 40 mM Tris, 500 mM NaCl, 1 Mmannopyranoside pH 7.4. A high-performance liquid chromatography system(Agilent Technologies) and MiniDAWN TREOS instrument (Wyatt) coupled toan Optilab T-rEX Refractive Index Detector (Wyatt) was used forperforming the SEC-MALS experiment. In total, either 100 μl of lectinelution or approximately 30 μg of protein was applied to a TSK-GelG3000SWxl column (Tosoh Bioscience) equilibrated in running buffer (150mM sodium phosphate, 50 mM NaCl, pH 7.0) at 1 mL/min. The data wereanalyzed using the Astra 6 software package, and molecular weightcalculations were derived from the refractive index signal.

The SEC-MALS chromatograms of the ConC_SOSIP envelope protein and theHIV gp140 variants containing single mutations are shown in FIG. 3 . Ingeneral, the results obtained from the SEC-MALS analysis were comparableto and consistent with the results obtained from the AlphaLISA analysis.The chromatogram of the ConC_SOSIP envelope protein has four majorpeaks, with the second peak that eluted at about 7.3 minutes being thetrimer peak. The ConC_SOSIP envelope protein was determined to be about27% trimeric. The formation of aggregates and monomers indicates thatthere is some misfolding and instability associated with HIV gp140 Envprotein having the ConC_SOSIP consensus sequence. As demonstrated by thechromatograms shown in FIG. 3 , all single substitutions resulted in arelatively higher trimer peak as compared to the trimer peak for theConC_SOSIP envelope protein, indicating that trimer yield was increasedfor each of the HIV gp140 variants.

Taken together, the results demonstrate that the amino acidsubstitutions identified in (i)-(vii) of Table 1 herein providerecombinant HIV Env proteins with improved percentage of trimerformation and/or improved trimer yield. In particular, HIV Env proteinvariants having multiple substitutions at the identified positions of(i)-(vii) of Table 1, such as combinations of two or more of theidentified mutations typically exhibited even more improved trimer yieldand/or percentage of trimer formation over HIV Env protein variantshaving only a single mutation, which shows a possible synergistic effectof combinations mutations (i)-(vii) of Table 1. HIV envelope proteinshaving an increased percentage of trimer formation are advantageous froma manufacturing perspective, such as for vaccines, because lesspurification and removal of the envelope protein present in thepreparation in the undesired non-native conformations will be required.Also, an increased total expression yield of the trimer is advantageousfor manufacturing a vaccine product.

Example 4: Recombinant HIV Envelope Protein Variants Based on a Clade BEnvelope Protein Consensus Sequence

Recombinant HIV Env proteins comprising a single amino acid substitution(I535N, D589V, N651F or K655I) introduced into the clade B consensussequence ConB_SOSIP (SEQ ID NO: 5) were generated and purified asdescribed in Example 2. The trimer yield and percentage of trimerformation were measured by AlphaLISA assay as described in Example 3.

The results are shown in FIG. 4A (percentage of trimer formation) andFIG. 4B (trimer yield). The values reported are relative to the valuemeasured for the ConB_SOSIP envelope protein, which was set to 1 forboth the percentage of trimer formation and trimer yield. The resultsshow that all of the mutations tested increased the percentage of trimerformation. The trimer yield was about the same or improved relative tothe ConB_SOSIP envelope protein for all of the mutations tested.

These results demonstrate that these mutations also had a stabilizingeffect on the envelope protein, e.g., improved trimer yield, improvedpercentage of trimer formation, etc., when introduced into a differentbackbone HIV envelope protein sequence, in this case a Clade B derivedconsensus sequence.

Example 5: Recombinant HIV Envelope Protein Variants Based on aSynthetic Envelope Protein Sequence

Recombinant HIV Env proteins comprising amino acid substitutionsintroduced into a synthetic HIV envelope protein (named‘DS_sC4_SOSIP_E166R’) having the sequence shown in SEQ ID NO: 7 wereprepared and purified as described in Example 2. The synthetic HIVenvelope protein DS_sC4_SOSIP_E166R has the so-called SOSIP mutations(Cys at residues 501 and 605, and Pro at residue 559), Cys at residues201 and 433 resulting in the introduction of a disulfide (DS) bond, andArg at position 166 to stabilize the apex. In addition, the protein istruncated at position 655. The percentage of trimer formation and trimeryield were measured by AlphaLISA assay as described in Example 3.

The results are shown in FIG. 5 , which compares the percentage oftrimer formation for each of the variants tested to the percentage oftrimer formation (FIG. 5A) and trimer yield (FIG. 5B) for theDS_sC4_SOSIP_E166R backbone. A greater percentage of trimer formationwas observed for each of the variants tested as compared to the backbonesequence.

Besides E166R, some other rarely occurring amino acids were changed intomore prevalent ones at the corresponding position in a collection ofwild-type HIV Env proteins (A114Q, E117K, T375S and I434M), to ‘repair’the protein according to a framework explained in more detail in example12 below and FIG. 13 . In this ‘repaired’ protein, the stabilizingmutations A204I, and K655I improve sC4_SOSIP even further (FIG. 14 ).

The results of this Example are consistent with those of Example 4 indemonstrating that the mutations described herein also have astabilizing effect on the envelope protein, e.g., improved percentage oftrimer formation, and/or improved trimer yield, when introduced intodifferent backbone HIV envelope protein sequences, in this case anon-consensus, synthetic, Env sequence.

Example 6: Further Combinations of HIV Env Mutations

Recombinant HIV Env proteins comprising amino acid substitutionsintroduced in ConC_SOSIP (having the sequence shown in SEQ ID NO: 3)were prepared and purified as described in Example 2. The percentage oftrimer formation was measured by AlphaLISA assay as described in Example3. Subsequently, a smaller selection of combinations (the ones depictedbelow in italic and additionally K655I; I535N, D589V; I535N, K655I;D589V, K655I) were purified using Galanthus nivalis lectin and trimercontent was analyzed using SEC-MALS as described in Example 3.

The following mutants were prepared for this experiment:

-   -   K655I, N651F;    -   K655I, N651F, E647F;    -   K655I, N651F, E647F, I535N;    -   K655I, N651F, 15351V;    -   K655I, I573F;    -   K655I, D589V, I573F;    -   K655I, D589V, I573F, N651F;    -   K655I, D589V, I573F, K588E;    -   K655I, D589V, I573F, N651F, K588E;    -   K655I, D589V, I573F, N651F, K588E, I535N;    -   K655I, D589V, I573F, N651F, K588E, I535N, A204I;    -   K655I, D589V, I535N, L556P;    -   K655I, D589V, I573F, N651F, K588E, L556P;    -   K655I, D589V, A204I;    -   L556P, N651F;    -   L556P, N651F, K655I;    -   L556P, N651F, K655I, I535N;    -   L556P, N651F, K655I, I535N, I573F;    -   L556P, N651F, K655I, I535N, I573F, D589V;    -   L556P, N651F, K655I, I535N, I573F, D589V, A204I;    -   L556P, N651F, K655I, I535N, I573F, D589V, A204I, K588Q;    -   L556P, N651F, K655I, I535N, I573F, D589V, A204I, K588Q, E647F;    -   L556P, N651F, I535N;    -   L556P, N651F, I535N, I573F;    -   L556P, N651F, I535N, I573F, D589V;    -   L556P, N651F, I535N, I573F, D589V, A204I;    -   L556P, N651F, I535N, I573F, D589V, A204I, K588Q;    -   L556P, N651F, I535N, I573F, D589V, A204I, K588Q, E647F;    -   L556P, K655I, I535N;    -   L556P, K655I, I535N, I573F;    -   L556P, K655I, I535N, I573F, D589V;    -   L556P, K655I, I535N, I573F, D589V, A204I;    -   L556P, K655I, I535N, I573F, D589V, A204I, K588Q;    -   L556P, K655I, I535N, I573F, D589V, A204I, K588Q, E647F;    -   L556P, N651F, I535N, I573F, D589V, A204I, K588Q with the SOS        mutation removed.

All tested combinations of substitutions in the ConC_SOSIP backboneshowed higher trimer percentage and higher trimer yield compared to thebackbone in AlphaLISA (data not shown). SEC MALS confirmed improvedtrimer percentage for all tested mutations in the backbone (data notshown).

For a set comprising one by one additional mutations up to ninemutations, SEC-MALS showed that by the introduction of each nextmutation the ratio trimer/monomer increased, as the height of themonomer peak decreased, while the height of the trimer peak stayed thesame in the SEC graph (FIG. 6 ). Of all the variants tested in SEC-MALS,the variant with L556P, N651F, K655I, I535N, I573F, D589V, A204I, K588Q,E647F substitutions showed the highest trimer percentage (the leastgp140 monomers and the least gp120 monomers), the highest total proteinyield and one of the higher temperature stabilities. This means thatthese mutations can be combined without loss of trimer compared to thebackbone. In addition, this suggests that, in general, addition ofmutations described in (i)-(vii) of Table 1, optionally combined withmutations described in Table 2, results in further improvedtrimerization.

A construct with the L556P, N651F, I535N, I573F, D589V, A204I, K588Qmutations wherein the ‘SOS mutations’ were removed (i.e. the twocysteine residues at positions 501 and 605 were reverted back into theamino acid residues that were originally present in the consensus cladeC sequence) was also tested. This mutant had comparable trimerpercentage and yield as its corresponding mutant that did comprise theSOS mutation. The mutant wherein the SOS mutation was removed even hadan advantage in that it bound less non-bNAbs than its correspondingSOS-containing counterpart (having the L556P, N651F, I535N, I573F,D589V, A204I, K588Q mutations). This demonstrates that advantageousproperties, such as high trimerization percentage, can also be obtainedin HIV Env proteins that do not have all the SOSIP mutations.

One mutant (tested in the ConC_SOSIP backbone), based upon a combinationof favorable properties in expression level, trimer formation andbinding to broadly neutralizing antibody PGT151, has the followingmutations: L556P, N651F, I535N, I573F, D589V, A204I, K588Q.

In the ConC_SOSIP background, the 9 most successful substitutions wereL556P, E647F, N651F, K655I, I535N, D589V, I573F, and K588E in gp41 andA204I in gp120. The combination of all these 9 substitutions led toincreased stability, trimer content and trimer yield. Since addition ofL556P in this variant with 9 substitutions had a relatively limitedeffect on improved trimer percentage, and the E647F substitution in thiscontext appeared to hamper PGT151 binding, these two mutations were notalways used in further variants, and a variant with 7 substitutions(named ConC_SOSIP 7mut, sometimes also referred to herein as ‘stabilizedConC_SOSIP’ or ‘ConC_base’; including N651F, K655I, I535N, D589V, I573F,K588E, and A204I) was found to be slightly more stable (increasedmelting temperature) than the variant with the 9 substitutions indicatedabove. The complete sequence of this variant (stabilized ConC_SOSIP Env,HIV 160544) is provided in SEQ ID NO: 20.

At this moment, a particularly preferred mutant [tested in theConC_SOSIP backbone with the following additional mutations: (a) D279N,A281V, A362Q (increase similarity to transmitted founder viruses, asdescribed by others); (b) Del139-152 (deletion of a variable loop toreduce chance of inducing antibodies to this loop); and (c) V295N(introduction of a glycan site that is present in the majority of HIVstrains)], based upon a combination of favorable properties inexpression level, trimer formation and binding to a broadly neutralizingantibody, has the following stabilizing mutations of the invention:N651F, K655I, I535N, I573F, D589V, A204I, K588E. The complete sequenceof this variant (Stabilized ConC_SOSIP.v3 Env (HIV170654,ConC_SOSIP.v3)) is provided in SEQ ID NO: 28.

In a further variant, a K658V mutation was added to this construct (seealso example 8 below), which further improved the results.

Example 7: Self-Assembling Particles Displaying Stabilized HIV EnvProtein

Ferritin and DPS self-assembling particles were prepared that displaystabilized Env proteins in a similar fashion as described in (He et al,2016). In order to do this the gp140 protein was fused to the N-terminusof the particles via a short amino acid linker (e.g. GSG or AAAGS, butother linkers can also be used, see e.g. He et al, 2016) at DNA leveland expressed the fusion protein in Expi293F cells. One example of aparticle that was prepared in this manner was based on ferritin fused toa ConC_SOSIP (SEQ ID NO: 3) HIV Env protein with the followingmutations: I535N, A558P, D589V, K655I. Ferritin particles with this Envprotein having an additional V570D mutation, which has been reported toimprove trimerization (Kesavardhana et al, 2014), were also prepared,but it was observed that this mutation leads to a strong increase inbinding of a non-neutralizing antibody (17b), which is undesired. Envwith these five mutations was also fused to two types of DPS particles,from Helicobacter pylori and from Mycobacterium smegmatis (see e.g.WO2011/082087 for preparation of DPS particles). Env with these fivemutations and in addition the disulfide bridge introducing doublemutation 1201C-A433C was also fused to ferritin.

The particles were purified from cell free supernatant with PGDM1400affinity beads and the particles were analyzed using SEC-MALS with aTSKgel G6000PWCL column. SEC-MALS, as well as Native PAGE (3-12%),confirmed that particles with approximately the expected sizes wereformed.

In a similar manner, ferritin and DPS self-assembling nanoparticlesdisplaying HIV Env having a ConC_SOSIP sequence with the followingcombination of mutations: (L556P, N651F, I535N, I573F, D589V, A204I,K588Q), are also prepared.

Further liposomes and/or self-assembling nanoparticles displaying otherHIV Env variants described herein, e.g. HIV Env having SEQ ID NO: 20,22, 24, 26, 27, 28, 29, 30, 31, or 32, are also prepared.

Ni-NTA liposomes and covalent click chemistry liposomes with some ofsuch variants were prepared (Ingale J, et al. Cell Rep. 2016,15(9):1986-99; Bak M, et al. Bioconjug Chem. 2016, 27(7):1673-80).Liposomes were analyzed using ns-TEM which showed evenly spaced,orthogonally displayed, and densely covered HIV Env protein on theliposome surface.

Example 8. HIV Env Protein with Trimer Stabilizing Mutation at Position658

Recombinant HIV Env proteins with substitution mutations at position 658(numbering according to gp160 of HIV-1 isolate HXB2) were prepared, inthe ConC_SOSIP (SEQ ID NO: 3) backbone. K658 was mutated into Val, Ile,Phe, Met, Ala, and Leu. In addition, some double mutants were madewherein these mutations were combined with one of the stabilizingmutations described above, K655I. The percentage of trimer formation wasdetermined by the AlphaLISA assay as described in Example 3.

The results are shown in FIGS. 7A and 7B (trimer percentage, measured indifferent experiments, hence two panels) and FIGS. 7C and 7D (trimeryield, measured in different experiments, hence two panels). Theseresults demonstrate that substitution at position 658 by Ile, Phe, Met,Leu, Ala, or Val resulted in improved percentage of trimer formation andimproved trimer yield. Substitution with Ile at position 658 resulted inincreases that are in about the same range as the K655I mutation (FIG.7A, C), which was the best performing single mutant from the mutationsin Table 1 described above (see e.g. FIG. 2A). Substitution with Val atposition 658 resulted in even higher improvement (FIG. 7A, C).

The results also demonstrated that substitution at position 658 by Ileor Val could be combined with mutation K655I that was described above,and that this resulted in a further improvement over each of thecorresponding single mutants (FIG. 7A, C).

The K658V mutant was also tested using SEC-MALS. 96-well cultures weregrown for three days as was done for the AlphaLISA. Supernatant wasdirectly loaded on a SEC-MALS column. The chromatograms obtained for themock supernatant (with furin expression) was subtracted from thechromatograms of the supernatant with Env proteins. The trimeric proteineluted from the column between 7 and 8 minutes. The results are shown inFIG. 8 , and confirmed that the K658V mutant showed improvedtrimerization over the background Env protein, and over the K655I mutantEnv protein.

This example demonstrates that substitution of the amino acid atposition 658 in HIV Env protein by Val, Ile, Phe, Met, Leu, or Ala,results in improved trimer percentage and trimer yield.

Further experiments to measure trimer formation of variants usingAlphaLISA and/or SEC-MALS are performed in HIV Env variants wherein theK658V mutation is present in combination with other mutations fromTables 1 and/or 2 as described herein, as well as in HIV strains fromclade A and B. For example, the 658V mutation has already been shown toimprove the ConC_SOSIP, 7mut variant as described above (example 7), aswell as BG505_SOSIP with L556P, K655I, M535N, N651F, D589V, K588E(described below in example 9), as well as the repaired and stabilizedC97ZA_SOSIP (described below in example 10).

Based on the results described above, it is expected that the mutationof the amino acid at position 658 into a Valine, Isoleucine,Phenylalanine, Leucine, Methionine or Alanine, preferably into a Valine,residue will improve trimer formation and/or trimer yield in differentbackground HIV Env proteins.

Example 9: Recombinant HIV Envelope Protein Variants Based on a Clade aEnvelope Protein Sequence

Single amino acid substitutions (I535N, D589V, N651F, K655I, I573F,A204I or E647F) were introduced into a wild type clade A HIV envelopeprotein with the SOSIP modification (named ‘BG505_SOSIP’) as describedin Example 2. The HIV envelope protein BG505_SOSIP has the so-calledSOSIP mutations (Cys at residues 501 and 605, and Pro at residue 559),as well as further Cys at residues 201 and 433 resulting in theintroduction of a disulfide (DS) bond, and a potential N-glycosylationsite on position 332 (T332N mutation). The protein is truncated atposition 664. The sequence of BG505_SOSIP is shown in SEQ ID NO: 21.

The percentage of trimer formation and trimer yield were measured byAlphaLISA assay as described in Example 3. The percentage of trimerformation and trimer yield for each of the variants tested was comparedto BG505_SOSIP. A higher percentage of trimer formation was observed forthe M535N, D589V, N651F or K655I substitutions as compared to thebackbone sequence (e.g. FIG. 9A). Combination of e.g. L556P, K655I andM535N showed an even more increased trimer yield and percentage (e.g.FIGS. 9A and 9B). Combination of N651F and D589V improved the trimeryield and percentage even more (data not shown). The results of thisExample for a clade A virus are consistent with those of examples 10 and11 (clade C) below and Example 5 (clade B), in which the mutationsI535N, D589V, N651F and K655I also showed a stabilizing effect on theenvelope protein derived from wild-type strains, e.g., improvedpercentage of trimer formation, and/or improved trimer yield. Clearly,these mutations also improve trimerization of HIV Env derived from awild-type clade A strain.

At this moment, a particularly preferred mutant (tested in theBG505_SOSIP backbone, based upon a combination of favorable propertiesin expression level, trimer formation and binding to a broadlyneutralizing antibody, is the one having the following mutations: L556P,K655I, M535N, N651F, D589V, (see e.g. FIG. 10 , showing a stronglyimproved trimer formation of such mutant in a SEC-MALS analysis, andFIG. 14 , showing a clearly improved binding of broadly neutralizingantibodies of such mutant). The sequence of this stabilized BG505_SOSIPEnv (HIV170863) is shown in SEQ ID NO: 22.

Addition of mutation Q658V provided a small further improvement.

A further preferred construct contains the L556P, K655I, M535N, N651F,D589V mutations, as well as the ‘DS’ mutations (Cys at positions 201 and433 resulting in introduction of a disulfide bond), R588E, and Q658V.The sequence of that variant (BG505_SOSIP.v2 Env, HIV171814) is providedin SEQ ID NO: 29.

Differential scanning calorimetry was used to determine meltingtemperatures, which are an indication of stability of HIV Env trimers.Melting temperatures for HIV Env were determined using MicroCalcapillary DSC system. 400 μL of 0.5 mg/mL protein sample was used permeasurement. The measurement was performed with a start temperature of20° C. and a final temperature of 110° C. The scan rate 100° C./h andthe feedback mode; Low (=signal amplification). The data were analyzedusing the Origin J. Software (MicroCal VP-analysis tool).

The melting temperature of the BG505_SOSIP.v2 Env (HIV171814) Envvariant (SEQ ID NO: 29) was measured with DSC to be 82.2° C., whereasthe BG505_SOSIP backbone (SEQ ID NO: 21) has a melting temperature of67.8° C.

Example 10: Recombinant HIV Envelope Protein Variants Based on Clade CWild Type Envelope Protein Sequence

Recombinant HIV Env proteins according to embodiments of the inventioncomprising the single amino acid substitution T651F, the double aminoacid substitution T651F, M535N introduced into a WT C97ZA_SOSIP Envsequence (SEQ ID NO: 23) with the additional substitution L556P(C97ZA_SOSIP_L556P) were generated and expressed as described in Example2. The trimer yield and percentage of trimer formation were measured byAlphaLISA assay as described in Example 3.

The results are shown in FIGS. 11A and B. The trimer yield ofC97ZA_SOSIP_L556P_T651F_M535N is five times higher than that of theC97ZA_SOSIP backbone.

The L556P, T651F and M535N substitutions thus gave a large improvementof C97ZA_SOSIP, but binding to bNAbs and trimer percentage for thisclade C wild-type derived variant was still much lower than for theConC_SOSIP backbone. Because a wt Env may be adapted to its host,possibly reducing its general fitness, and thereby the folding may becorrupted, the Env sequence was ‘repaired’ according to the conceptualframework described below in Example 12 and in FIG. 13 . A total of 21residues were changed, to repair the sequence, and three potentialN-glycosylation sites (PNGS) were added to fill the so-called “glycanholes” (positions where in at least 50% of the wild-type HIV strains Envprotein a potential N-glycosylation site is present). The mutationsintroduced by following this framework for C97ZA_SOSIP are indicated inTable 3 in the column ‘repairing mutations’. Addition of stabilizingmutation K655I disclosed herein increased the trimer percentage andyield even further, as did D589V, A204I and K588E.

These results demonstrate that the T651F, M535N and K655I, D589V, A204Iand K588E mutations described herein also had a stabilizing effect onthe envelope protein, e.g., improved trimer yield, improved percentageof trimer formation when introduced into C97ZA_SOSIP (derived from aclade C wild-type strain Env protein) and variants thereof.

At this moment, a particularly preferred variant (tested in theC97ZA_SOSIP backbone), based upon a combination of favorable propertiesin expression level, trimer formation and binding to a broadlyneutralizing antibody, is the one having the following mutations: Q567K(described by others before); A198T, S243N, K236T, V295N (to fill glycanholes); M34L, T46K, T58A, Q171K, G172V, P179L, L183Q, I192R, N209T,M3071, Q350R, N352H, Y353F, D412N, G429E, V455T, I489V, L4911, G500K,S547G, T578A, T651N (to repair the sequence); V505N, E507T, T663N (addedpotential N-glycosylation sites at base of molecule); and A204I, M535N,L556P, K588E, D589V, T651F, K655I (stabilizing mutations of invention).Data for this variant are for instance shown in FIG. 14 , see inparticular ‘stabilized and repaired C97ZA’ therein), showing a hugeincrease in broadly neutralizing antibody binding as compared to theoriginal wt C97ZA Env molecule. The sequence of this variant (stabilizedand repaired C97ZA_SOSIP Env (HIV170690)) is provided in SEQ ID NO: 24.

Addition of mutation K658V stabilized this protein even further.

A further preferred variant includes the ‘DS’ mutation and K658V, andthe sequence of this variant (repaired and stabilized C97ZA_SOSIP.v2Env, HIV171810) is provided in SEQ ID NO: 30. The melting temperature ofthis protein is 80.2° C., determined by DSC.

Example 11: Recombinant HIV Envelope Protein Variants Based on AnotherClade C Wild Type Envelope Protein Sequence

In the Env protein from clade C strain Du422, SOSIP mutations wereintroduced and two glycan holes were filled at position 295 and 386 byK295N and D386N mutations. In addition, some residues were repairedaccording to the conceptual framework described in Example 12 and FIG.13 (V272I, W456R, G466E and F643Y), and stabilizing substitutions L556P,I535N, N651F and D589V were introduced. All additional substitutionsresulted in higher trimer yields and trimer percentages (e.g. FIG. 12 ).

In a specific tested variant with these four stabilizing mutations (SEQID NO: 25), the additional K655I substitution further increased trimeryield and trimer percentage by a factor 1.3 and 1.4 respectively (datanot shown).

At this moment, a particularly preferred Du422_SOSIP Env variant, basedupon a combination of favorable properties in expression level, trimerformation and binding to a broadly neutralizing antibody, is the onehaving the following mutations: L556P, K655I, M535N, N651F, D589V,K588E, I201C, A433C, V272I, W456R, G466E, F643Y, D386N, and K295N. Thesequence of this variant (stabilized and repaired Du422_SOSIP Env(HIV170859) is provided in SEQ ID NO: 26. Data for this variant are forinstance shown in FIG. 14 (see stabilized and repaired Du422 therein),showing a huge increase in broadly neutralizing antibody bindingcompared to the original wt Du422 Env molecule.

A further preferred variant additionally comprises the ‘DS’ mutation andK658V, and the sequence of this variant (repaired and stabilizedDu422_SOSIP.v1 Env, HIV171812) is provided in SEQ ID NO: 31. The meltingtemperature of this protein is 78.9° C., determined by DSC.

Example 12: Repairing and Stabilizing Various HIV-1 Env Sequences

Because wt sequences from viruses isolated from infected patients mayhave acquired destabilizing mutations that impede correct folding, wtEnv sequences of clade C C97ZA, DU422 and the mosaic sC4 were firstrepaired.

To search for non-optimal mutations in wild type sequences an alignmentof all HIV-1 Env sequences in the UniProt database and the Los AlamosHIV database (˜90.000 sequences) was made and the amino aciddistribution was calculated for each amino acid. In general, a number ofrelatively rarely occurring amino acids in wt Env sequences weresubstituted into more common amino acids (based upon frequency in thedatabase at the corresponding position) according to the conceptualframework described in FIG. 13 .

Furthermore, two additional substitutions Y353F and Q171K at the apex ofC97ZA_SOSIP were introduced to possibly improve the binding of apextargeting antibodies, and extra glycan sites were introduced by thesubstitution of D411N, K236T and V295N because these potentialN-glycosylation sites (PNGS) were conserved >50%. Next, stabilizingsubstitutions described in previous examples were transferred to therepaired sequence.

The stabilized ConC_SOSIP contains the substitutions A204I, I535N,I573F, K588E, D589V, N651F and K655I (stabilized ConC_SOSIP). Thecomplete sequence of stabilized ConC_SOSIP is provided in SEQ ID NO: 20.

An overview of some of the variant Env proteins and their mutations isprovided in Table 3.

TABLE 3 HIV Env protein variants. leader mutations sequence from added(SEQ ID repairing stabilizing Protein literature PNGS NO:) mutationsmutations other mutations terminus ConC_SOSIP A501C, V295N 11 664 T605C,I559P Stabilized A501C, V295N 11 A204I, 664 ConC_SOSIP T605C, I535N,I559P I573F, K588E, D589V, N651F, K655I Stabilized A501C, V295N 11A204I, delta138-152, 664 ConC_SOSIP.v3 T605C, I535N, D297N, A281V, I559PI573F, A362Q K588E, D589V, N651F, K655I BG505_SOSIP A501C, T332N 34 664T605C, I559P stabilized A501C, T332N 34 M535N, 664 BG505_SOSIP T605C,L556P, I559P D589V, N651F, K655I stabilized A501C, T322N 34 M535N, 664BG505_SOSIP.v2 T605C, L556P, I559P, D589V, I201C, N651F, A433C K655I,R588E, Q658V C97ZA_SOSIP A501C, 43 664 T605C, I559P L535M, Q567Krepaired A501C, A198T, 11 M34L, T46K, V505N, E507T, 664 C97ZA_SOSIPT605C, S243N, T58A, T663N I559P, K236T, Q171K, L535M, V295N G172V, Q567KP179L, L183Q, I192R, N209T, M307I, Q350R, N352H, Y353F, D412N, G429E,V455T, I489V, L491I, G500K, S547G, T578A, T651N repaired and A501C,A198T, 11 M34L, T46K, A204I, V505N, E507T, 664 stabilized T605C, S243N,T58A, M535N, T663N C97ZA_SOSIP I559P, K236T, Q171K, L556P, Q567K V295NG172V, K588E, P179L, D589V, L183Q, T651F, I192R, K655I N209T, M307I,Q350R, N352H, Y353F, D412N, G429E, V455T, I489V, L491I, G500K, S547G,T578A repaired and A501C, A198T, 11 M34L, T46K, A204I, V505N, E507T, 664stabilized T605C, S243N, T58A, M535N, T663N C97ZA_SOSIP.v2 I559P, K236T,Q171K, L556P, Q567K, V295N G172V, K588E, I201C, P179L, D589V, A433CL183Q, T651F, I192R, K655I, N209T, K658V M307I, Q350R, N352H, Y353F,D412N, G429E, V455T, I489V, L491I, G500K, S547G, T578A Du422_SOSIPA501C, D386N, 11 664 T605C, K295N I559P repaired A501C, D386N, 11 V272I,664 Du422_SOSIP T605C, K295N W456R, I559P G466E, F643Y repaired andA501C, D386N, 11 V272I, M535N, 664 stabilized T605C, K295N W456R, L556P,Du422_SOSIP I559P G466E, K588E, F643Y D589V, N651F, K655I repaired andA501C, D386N, 11 V272I, M535N, — 664 stabilized T605C, K295N W456R,L556P, Du422_SOSIP.v1 I559P, G466E, K588E, I201C, F643Y D589V, A433CN651F, K655I, K658V DS_sC4_SOSIP A501C, V295N 33 655 T605C, I559P,I201C, A433C repaired A501C, V295N 33 A114Q, 655 DS_sC4_SOSIP T605C,E117K, I559P, E166R, I201C, T375S, A433C I434M repaired and A501C, V295N33 A114Q, A204I, deltal38-152 655 stabilized T605C, E117K, I535N,(SSNGTYNIIHN DS_sC4_SOSIP I559P, E166R, L556P, ETYK), delta191 I201C,T375S, Q588E, (SEKSSENSSE), A433C I434M D589V, delta 463 (GVP) N651F,K655I repaired and A501C, V295N 33 A114Q, A204I, delta138-152 655stabilized T605C, E117K, I535N, (SSNGTYNIIHN DS_sC4_SOSIP.v4 I559P,E166R, L556P, ETYK), delta191 I201C, T375S, Q588E, (SEKSSENSSE), A433CI434M N651F, delta 463 (GVP) K655I repaired A501C, — 42 S72H, — — 664ADM30337.1 T605C, D234N, I559P R651N, F602L, Y621D, V413T, V316T, V544Lrepaired and A501C, — 42 S72H, I535N, — 664 stabilized T605C, D234N,L556P, ADM30337.1 I559P R651N, K588E, F602L, D589V, Y621D, N651F, V413T,K655I, V316T, K658V V544L repaired A501C, — 39 S67N, I156N, — — 664ZM233M T605C, V174A, I559P V414I, M33N, T347K, H638Y, A394T, F274S,T471G, S316T, V323I, L410S, Y134V, A335E, I395Y, A65V, D130N, A612S,I111L, S195N, N477D, K152E, L181I, S463N repaired and A501C, — 39 S67N,I156N, M535N, — 664 stabilized T605C, V174A, L556P, ZM233M I559P V414I,K588E, M33N, D589V, T347K, N651F, H638Y, K655I, A394T, K658V F274S,T471G, S316T, V323I, L410S, Y134V, A335E, I395Y, A65V, D130N, A612S,I111L, S195N, N477D, K152E, L181I, S463N repaired A501C, — 40 Y187bN, —— 664 CN97001 T605C, A77T, I232T, I559P V33N, Q354P, E99D, P462N, T185N,T49K, Q105H, N102D, V525A, R132T, E130N, V164E, N477D, T219A repairedand A501C, — 40 Y187bN, I535N, — 664 stabilized T605C, A77T, I232T,L556P, CN97001 I559P V33N, K588E, Q354P, D589V, E99D, N651F, P462N,K655I, T185N, K658V T49K, Q105H, N102D, V525A, R132T, E130N, V164E,N477D, T219A repaired A501C, — 41 S113D, — — 664 ZM246F T605C, R339N,I559P P63K, K415T, K172E, T153E, S335E, S160N, I303T, K448N, I444T,G347K, Q106E, G293E, I135N repaired and A501C, — 41 S113D, I535N, — 664stabilized T605C, R339N, L556P, ZM246F I559P P63K, K588E, K415T, D589V,K172E, N651F, T153E, K655I, S335E, K658V S160N, I303T, K448N, I444T,G347K, Q106E, G293E, I135N

Table 3. Several of HIV Env protein variants described herein. Thecolumn ‘mutations from literature’ describes mutations that were used inthese constructs and previously described by others. The column ‘addedPNGS’ describes mutations that add a potential N-glycosylation site (atpositions where many wild type Env proteins comprise such a site). Thecolumn ‘leader sequence’ describes which leader sequence was used forexpression if it was not the original (native) leader sequence. Thecolumn ‘repairing mutations’ describes the mutations that improvefolding and stability (measured as trimer yield and percentage, based onbinding to bNAbs) of some of the wild-type Env proteins, as described inExample 12 and FIG. 13 . The column ‘stabilizing mutations’ describesmutations from Tables 1 and 2 that stabilize the protein and improvetrimerization as disclosed herein. The column ‘further mutations’describes additional mutations made for some constructs. The column‘terminus’ describes the position of the last amino acid (numberingthroughout the table is with respect to HXB2 Env sequence).

Supernatants of cells transiently transfected with wild-type (wt),repaired, and stabilized Env variants were tested for binding to severaltrimer-specific broadly neutralizing antibodies directed to the apex.The repair substitutions and especially the stabilizing substitutionshad a dramatic impact on trimer content (FIGS. 14 and 15 ), determinedwith AlphaLISA (FIG. 14 ) and SEC-MALS (FIG. 15 ).

The sequence of a preferred variant of the repaired and stabilizedDS_sC4 Env protein (repaired and stabilized DS_sC4_SOSIP Env(HIV170686)) is provided in SEQ ID NO: 27.

Another preferred variant thereof is provided in SEQ ID NO: 32 (repairedand stabilized sC4_SOSIP.v4 Env). The melting temperature of thisprotein is 82.8° C., determined with DSC.

Example 13: Stabilizing Mutations of the Invention Function in theAbsence of the SOSIP Mutations

As shown in previous examples, the 7 mutations (A204I, I535N, I573F,K588E, D589V, N651F and K655I) improved the trimer yield and percentagein the ConC_SOSIP (resulting in ‘ConC_base’ or ‘stabilized ConC_SOSIP’or ‘ConC_SOSIP 7mut’) (e.g. FIGS. 14 and 16 ).

This example demonstrates that the different SOSIP mutations (i.e. the‘SOS’ mutation: 2 substitutions by Cys residues at positions 501 and605; and the ‘IP mutation’: substitution by Pro residue at position 559)contribute to further stabilization, but are not required to obtainbenefits from the mutations of the invention.

The 7 mutations were shown to also improve trimer yield in the so-calledConC_SOS, which does not contain the stabilizing I559P mutation (‘IP’mutation), as shown in FIG. 16 (compare ConC_SOS vs ConC_SOS, 7mut).Hence, the ‘IP’ mutation is not essential for obtaining a benefit fromthe mutations described herein. Addition of the I559P mutation resultedin a big increase, showing that the ‘IP’ mutation is beneficial in thisconstruct in addition to the 7 mutations of the invention. Thestabilizing IP mutation (I559P) could also be replaced by A558P orL556P, both of these also resulting in a big increase over the variantlacking the I559P mutation.

Also the ConC IP, 7 mut, which contains the 7 mutations of the inventiondescribed above, but lacks the ‘SOS’ mutations, still showed a very hightrimer yield, demonstrating that also the ‘SOS’ mutations are notessential for obtaining benefit from the mutations described herein(e.g. compare ConC_SOSIP vs ConC IP, 7 mut), in line with observationsin example 7. Addition of the ‘SOS’ mutation does further increase thetrimer yield.

Thus, while Env trimers containing the stabilizing mutations describedherein can benefit from further stabilization with the SOSIP mutations,none of the 3 SOSIP mutations is required for obtaining benefits (e.g.improved trimer yield) of the stabilizing mutations described herein.

Example 14: Methionine Substitution at Positions 647, 651 or 655Improves Trimer Quality

Further to the mutations described in example 2, positions 589, 647, 651and 655 were individually substituted by a Met residue in a ConC_SOSIP(SEQ ID NO: 3) backbone, and tested for trimerization percentage andyield using methods as described above. It was shown that a Met atpositions 647, 651, or 655, like the mutations described in example 2,improved the quality of the trimer (higher trimer percentage and yield,increased bNAb binding), as can be seen in FIG. 17 .

Thus, apart from substitution by Phe, Ala, or Trp at position 651,substitution by Met at position 651 also improves trimer formation;apart from substitution by Phe, Ile, or Trp at position 655,substitution by Met at position 655 also improves trimer formation; andapart from substitution by Phe, or Ile at position 647, substitution byMet at position 647 also improves trimer formation.

Example 15. Immunization with Stabilized HIV Env Proteins

A rabbit immunization study was conducted with Env proteins coupled toliposomes as described in example 7. The prime is performed withstabilized ConC_SOSIP.v3 (SEQ ID NO: 28) displayed on Ni-NTA liposomes,and followed by four boosts with covalent click liposomes, each withanother protein, i.e. with 1) repaired and stabilized sC4_SOSIP.v4 (SEQID NO: 32); 2) repaired and stabilized C97ZA_SOSIP.v2 (SEQ ID NO: 30);3) repaired and stabilized Du422_SOSIP.v1 (SEQ ID NO: 31); and 4)stabilized BG505_SOSIP.v2 (SEQ ID NO: 29).

Serum is isolated after successive immunizations, and analyzed forinduced antibodies that particularly bind to the stable, closed,pre-fusion conformation of Env (using ELISA), as well as for inductionof bNAbs (using virus neutralization assays).

So far, serum was isolated after prime and boosts 1, 2 and 3, andanalyzed for induction of heterologous Tier 2 neutralizing Abs usingvirus neutralization assays. Data are shown in FIG. 19 . Neutralizingactivity was observed in sera of 7/7 animals against at least 2different heterologous Tier 2 clade C pseudoviruses, indicative oflimited heterologous Tier 2 NAb induction in Env-immunized animalscompared with sham-injected control animals. To date only few researchgroups have reported comparable levels of heterologous Tier 2neutralization in rabbit immunogenicity models.

Example 16. Repair and Stabilization of Several Wt Clade C HIV EnvProteins that are Known to Form Very Low Levels of Trimers

In the Env protein derived from clade C strain ZM233M, the worst foldingEnv known from the literature (Julien et al, 2015, supra), SOSIPmutations were introduced. In addition, a number of residues wererepaired according to the conceptual framework described in Example 12and FIG. 13 (S67N, I156N, V174A, V414I, M33N, T347K, H638Y, A394T,F274S, T471G, S316T, V323I, L410S, Y134V, A335E, I395Y, A65V, D130N,A612S, I111L, S195N, N477D, K152E, L181I, S463N), and stabilizingsubstitutions M535N, L556P, K588E, D589V, N651F, K655I, K658V wereintroduced. The sequence of this variant (stabilized and repaired ZM233MEnv (HIV172520) is provided in SEQ ID NO: 35. Data for this variant arefor instance shown in FIG. 14 (see stabilized and repaired ZM233Mtherein), showing a very high increase in broadly neutralizing antibodybinding compared to the original wt ZM233M SOSIP Env molecule.

In the Env protein derived from clade C strain CN97001, SOSIP mutationswere introduced. In addition, a number of residues were repairedaccording to the conceptual framework described in Example 12 and FIG.13 (Y187bN, A77T, I232T, V33N, Q354P, E99D, P462N, T185N, T49K, Q105H,N102D, V525A, R132T, E130N, V164E, N477D, T219A; note: position 187b isa position not present in HXB2: for such residues, typically letters areadded for any inserted amino acid residues behind the last amino acidthat corresponds to a HXB2 residue, e.g. 187a, 187b, etc), andstabilizing substitutions M535N, L556P, K588E, D589V, N651F, K655I,K658V were introduced. The sequence of this variant (stabilized andrepaired Env CN97001 (HIV172523) is provided in SEQ ID NO: 36. Data forthis variant are for instance shown in FIG. 14 (see stabilized andrepaired CN97001 therein), showing a very high increase in broadlyneutralizing antibody binding compared to the original wt CN97001 SOSIPEnv molecule.

In the Env protein derived from clade C strain ZM246F, SOSIP mutationswere introduced. In addition, a number of residues were repairedaccording to the conceptual framework described in Example 12 and FIG.13 (S113D, R339N, P63K, K415T, K172E, T153E, S335E, 5160N, I303T, K448N,I444T, G347K, Q106E, G293E, I135N), and stabilizing substitutions M535N,L556P, K588E, D589V, N651F, K655I, K658V were introduced. The sequenceof this variant (stabilized and repaired Env ZM246F (HIV172526) isprovided in SEQ ID NO: 37. Data for this variant are for instance shownin FIG. 14 (see stabilized and repaired ZM246F therein), showing a veryhigh increase in broadly neutralizing antibody binding compared to theoriginal wt ZM246F SOSIP Env molecule.

In the Env protein from clade C strain with Genbank accession numberADM30337.1, SOSIP mutations were introduced. In addition, a number ofresidues were repaired according to the conceptual framework describedin Example 12 and FIG. 13 (S72H, D234N, R651N, F602L, Y621D, V413T,V316T, V544L), and stabilizing substitutions M535N, L556P, K588E, D589V,N651F, K655I, K658V were introduced. The sequence of this variant(stabilized and repaired Env ADM30337.1 (HIV172517) is provided in SEQID NO: 38. Data for this variant are for instance shown in FIG. 14 (seestabilized and repaired ADM30337.1 therein), showing a very highincrease in broadly neutralizing antibody binding compared to theoriginal wt ADM30337.1 SOSIP Env molecule.

This example demonstrates that the methods described herein can be usedfor a wide variety of different HIV Env proteins, including proteinsthat were known to have very low (i.e. amongst the lowest reported)trimerization levels, and that the trimerization levels of such proteinscan also be dramatically improved, showing the general applicability ofthe methods and substitutions disclosed herein for improvingtrimerization of HIV Env proteins.

Example 17. Stabilization of a Clade B Strain with Mutation at Position658

In ConB_SOSIP (SEQ ID NO: 5) the Q658V substitution increased the trimeryield in analytical SEC using cell culture supernatants aftertransfection (FIG. 18 ), demonstrating that 658V increases trimer yieldof clade B, besides the observed trimer yield increases in clade C Envand a clade A Env described in previous examples.

Example 18. Stabilizing Mutations in Membrane-Bound Consensus C SOSIP

HEK293F cells were transfected with DNA constructs expressingmembrane-bound full-length (FL) Consensus C (ConC) SOSIP (SEQ ID NO:44), either with or without stabilizing amino acid substitutions A204I,I535N, I573F, K588E, D589V, N651F, and K655I. Two days posttransfection, cells were incubated with a panel of broadly neutralizingand non-broadly neutralizing antibodies (bNAbs and non-bNAbs) anddetected with Alexa Fluor 647 (AF647)-labeled anti-human secondaryantibody using fluorescence activated cell sorting (FACS). Theintegrated median fluorescence intensity (iMFI) was calculated bymultiplying the frequency of AF647 positive cells by the MFI, thusproviding a metric that incorporates both the magnitude and quality of aresponse (e.g., Darrah P A, et al. Nat Med. 2007, 13(7): 843-50). Dataare represented as fold-change over the backbone construct withoutstabilizing mutations (ConC_SOSIP_FL) in FIG. 20 . Overall, the effectof stabilizing mutations on bNAb iMFI in the membrane-bound contextappears limited, with 1 out of 10 bNAbs showing a higher, and 2 out of10 bNAbs showing a lower signal compared to the backbone. However, all 8non-bNAbs show a reduction of iMFI in the presence of stabilizingmutations, demonstrating the beneficial effect of these substitutions inthe membrane-bound context.

Nucleic acid sequences encoding membrane-bound stabilized ConC_SOSIP Envvariants were cloned into adenovirus (serotype 26) vectors. Adenovirusvectors encoding membrane-bound stabilized Env variants of the inventioncan also be used for vaccination.

The examples above demonstrate that the invention provides a universalapproach to optimize the folding and stability of prefusion-closed HIVenvelope trimer proteins.

It is understood that the examples and embodiments described herein arefor illustrative purposes only, and that changes could be made to theembodiments described above without departing from the broad inventiveconcept thereof. It is understood, therefore, that this invention is notlimited to the particular embodiments disclosed, but it is intended tocover modifications within the spirit and scope of the invention asdefined by the appended claims.

REFERENCES

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LIST OF SEQUENCESSEQ ID NO: 1 gp160 of HIV-1 isolate HXB2 (signal sequence in italics; aminoacids at positions (i)-(vii) of Table 1 indicated by grey shading; amino acid at position 658 underlined and bold)MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIME

LKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGA

TNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILLSEQ ID NO: 2 HIV Env consensus clade C (consensus sequence only, not including any signal sequence, transmembrane domain (664 is last amino acid), SOSIP mutations, and/or furin cleavage site mutations; amino acids at positions (i)- (vii) of Table 1 indicated by grey shading; amino acid at position 658underlined and bold)NLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTNNNNMKEEMKNCSFNTTTEIRDKKQKEYALFYRLDIVPLNENS

SLAEEEIIIRSENLTDNAKTIIVHLNESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFPNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTK

SEQ ID NO: 3 ConC_SOSIP (mature clade C consensus sequence with SOSIP mutationsand furin cleavage site (in italics), and C-terminal truncation; amino acidsat positions (i)-(vii) of Table 1 indicated by grey shading; amino acid atposition 658 underlined and bold)NLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTNNNNMKEEMKNCSFNTTTEIRDKKQKEYALFYRLDIVPLNENS

SLAEEEIIIRSENLTDNAKTIIVHLNESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFPNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTK

SEQ ID NO: 4 HIV Env consensus clade B (consensus sequence only, not includingany signal sequence, transmembrane domain (664 is last amino acid), SOSIPmutations, and/or furin cleavage site mutations; amino acids at positions (i)-(vii) of Table 1 indicated by grey shading; amino acid at position 658underlined and bold)AEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDLNNNTTNNNSSSEKMEKGEIKNCSFNITTSIRDKVQKEYALFYKLDV

TQLLLNGSLAEEEVVIRSENFTDNAKTIIVQLNESVEINCTRPNNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNISRTKWNNTLKQIVKKLREQFGNKTIVFNQSSGGDPEIVMHSFNCGGEFFYCNTTQLFNSTWNSNGTWNNTTGNDTITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNNNNNTTETFRPGGGDMRDNWRSELYKY

SEQ ID NO: 5 ConB_SOSIP (mature clade B consensus sequence with SOSIP mutationsand furin cleavage site (in italics), and C-terminal truncation; amino acidsat positions (i)-(vii) of Table 1 indicated by grey shading; amino acid atposition 658 underlined and bold)AEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDLNNNTTNNNSSSEKMEKGEIKNCSFNITTSIRDKVQKEYALFYKLDV

TQLLLNGSLAEEEVVIRSENFTDNAKTIIVQLNESVEINCTRPNNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNISRTKWNNTLKQIVKKLREQFGNKTIVFNQSSGGDPEIVMHSFNCGGEFFYCNTTQLFNSTWNSNGTWNNTTGNDTITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNNNNNTTETFRPGGGDMRDNWRSELYKY

SEQ ID NO: 6 synthetic HIV envelope protein Mos2S Env C4 fragment; amino acidsat positions (i)-(vii) of Table 1 indicated by grey shading)MGNLWVTVYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDASLEPCVKLTPLCVTLNCRNVRNVSSNGTYNIIHNETYKEMKNCSFNATTVVEDRKQKVHALFYRLD

QCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNITCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTIKFAPHSGGDLEITTHTFNCRGEFFYCNTSNLFNESNIERNDSIITLPCRIKQIINMWQEVGRAIYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGGDMRNNWR

SEQ ID NO: 7 (DS_sC4_SOSIP_E166R sequence; amino acids at positions (i)-(vii)of Table 1 indicated by grey shading)MGNLWVTVYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDASLEPCVKLTPLCVTLNCRNVRNVSSNGTYNIIHNETYKEMKNCSFNATTVVRDRKQKVHALFYRLD

QCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTIKFAPHSGGDLEITTHTFNCRGEFFYCNTSNLFNESNIERNDSIITLPCRIKQIINMWQEVGRCIYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGGDMRNNWR

SEQ ID NO: 8 (Mos1.Env, mosaic HIV envelope protein sequence; amino acids atpositions (i)-(vii) of Table 1 indicated by grey shading)AGKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDDVRNVTNNATNTNSSWGEPMEKGEIKNCSFNITTSIRNKVQKQYALF

GIRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIMVQLNVSVEINCTRPNNNTRKSIHIGPGRAFYTAGDIIGDIRQAHCNISRANWNNTLRQIVEKLGKQFGNNKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTKLFNSTWTWNNSTWNNTKRSNDTEEHITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNDTSGTEIFRPGGGD

SEQ ID NO: 9 (Mos2.Env, mosaic HIV envelope protein sequence; amino acids atpositions (i)-(vii) of Table 1 indicated by grey shading)MGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIIRLWDQSLKPCVKLTPLCVTLECRNVRNVSSNGTYNIIHNETYKEMKNCSFNATTVVEDRKQKVHALFYRLD

QCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNITCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTINFTSSSGGDLEITTHSFNCRGEFFYCNTSGLFNGTYMPNGTNSNSSSNITLPCRIKQIINMWQEVGRAMYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGGDM

SEQ ID NO: 10 (furin cleavage site mutant sequence) RRRRRRSEQ ID NO: 11 (example of a signal sequence (e.g. used for ConC_SOSIP, andsome wild-type derived variants))MRVRGILRNWQQWWIWGILGFWMLMICNVVG (note: the last VG could be the beginning ofthe mature protein or the end of the signal sequence)SEQ ID NO: 12 (example of 8 amino acid sequence that can replace HR1 loop)NPDWLPDMSEQ ID NO: 13 (example of 8 amino acid sequence that can replace HR1 loop)GSGSGSGSSEQ ID NO: 14 (example of 8 amino acid sequence that can replace HR1 loop)DDVHPDWDSEQ ID NO: 15 (example of 8 amino acid sequence that can replace HR1 loop)RDTFALMMSEQ ID NO: 16 (example of 8 amino acid sequence that can replace HR1 loop)DEEKVMDFSEQ ID NO: 17 (example of 8 amino acid sequence that can replace HR1 loop)DEDPHWDPSEQ ID NO: 18 (example of a signal sequence (e.g. used for ConB_SOSIP)MRVKGIRKNYQHLWRWGTMLLGMLMICSASEQ ID NO: 19 (tag used for HIV gp140 constructs in AlphaLISA assay)AAALPETGGGSDYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHHSEQ ID NO: 20 (stabilized ConC_SOSIP, ‘ConC_SOSIP_7mut’ (HIV160544))NLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTNNNNMKEEMKNCSFNTTTEIRDKKQKEYALFYRLDIVPLNENSSEYRLINCNTSTITQiCPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTDNAKTIIVHLNESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFPNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTKCKRRVVERRRRRRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLLRAPEAQQHMLQLTVWGfKQLQARVLAIERYLevQQLLGIWGCSGKLICCTAVPWNSSWSNKSQEDIWDNMTWMQWDREISNYTDTIYRLLEESQfQQEiNEKDLLALD SEQ ID NO: 21 (BG505_SOSIP Env protein (HIV150673))AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDSEQ ID NO: 22 (stabilized BG505_SOSIP Env protein (HIV170863))AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARNLLSGIVQQQSNLpRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRvQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQfQQEiNEQDLLALDSEQ ID NO: 23 (wtC97ZA_SOSIP Env protein with L535M and Q567K (HIV150673))NMWVTVYYGVPVWTDAKTTLFCASDTKAYDREVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNATFKNNVTNDMNKEIRNCSFNTTTEIRDKKQQGYALFYRPDIVLLKENRNNSNNSEYILINCNASTITQACPKVNFDPIPIHYCAPAGYAILKCNNKTFSGKGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEKEIIIRSENLTDNVKTIIVHLNKSVEIVCTRPNNNTRKSMRIGPGQTFYATGDIIGDIRQAYCNISGSKWNETLKRVKEKLQENYNNNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTTRLFNNNATEDETITLPCRIKQIINMWQGVGRAMYAPPIAGNITCKSNITGLLLVRDGGEDNKTEEIFRPGGGNMKDNWRSELYKYKVIELKPLGIAPTGcKRRVVERrrrrRAVGIGAVFLGFLGAAGSTMGAASmTLTVQARQLLSSIVQQQSNLLRApEAQQHMLkLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKLICcTNVPWNSSWSNKSQTDIWNNMTWMEWDREISNYTDTIYRLLEDSQTQQEKNEKDLLALDSEQ ID NO: 24 (repaired and stabilized C97ZA_SOSIP Env protein (HIV170690))NlWVTVYYGVPVWkDAKTTLFCASDaKAYDREVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNATEKNNVTNDMNKEIRNCSFNTTTEIRDKKQkvYALFYRlDIVqLKENRNNSNNSEYrLINCNtSTITQiCPKVtFDPIPIHYCAPAGYAILKCNNKTFnGtGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEKEIIIRSENLTDNVKTIIVHLNKSVEInCTRPNNNTRKSiRIGPGQTFYATGDIIGDIRQAYCNISGSKWNETLKRVKEKLrEhfNNNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTTRLFNNNATEnETITLPCRIKQIINMWQeVGRAMYAPPIAGNITCKSNITGLLLtRDGGEDNKTEEIFRPGGGNMKDNWRSELYKYKVvEiKPLGIAPTkcKRRnVtRrrrrRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARQLLSgIVQQQSNLpRApEAQQHMLkLTVWGIKQLQaRVLAIERYLevQQLLGIWGCSGKLICcTNVPWNSSWSNKSQTDIWNNMTWMEWDREISNYTDTIYRLLEDSQfQQEiNEKDLLAnDSEQ ID NO: 25 (variant of repaired and stabilized Du422 construct (HIV161818))NLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCKNVNISANANATATLNSSMNGEIKNCSFNTTTELRDKKQKVYALFYKPDVVPLNGGEHNETGEYILINCNSSTcTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNIKTIIVHLNKSVEINCTRPNNNTRKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQVKEKLREHYNKTIKFEPSSGGDLEVTTHSFNCRGEFFYCNTTKLFNETKLFNESEYVDNKTIILPCRIKQIINMWQEVGRcMYAPPIEGNITCKSNITGLLLTRDGGENSTEEVFRPGGGNMKDNWRSELYKYKVVEIKPLGVAPTKCKRKnVtRRRRRRAVGLGAVLLGFLGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLpRAPEAQQHLLQLTVWGIKQLQTRVLAIERYLKvQQLLGLWGCSGKLICCTAVPWNSSWSNKSLGDIWDNMTWMQWDREISNYTNTIYRLLEDSQfQQEKNEKDLLAnDSEQ ID NO: 26 (repaired and stabilized Du422_SOSIP (HIV170859))NLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCKNVNISANANATATLNSSMNGEIKNCSFNTTTELRDKKQKVYALFYKPDVVPLNGGEHNETGEYILINCNSSTITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNIKTIIVHLNKSVEINCTRPNNNTRKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQVKEKLREHYNKTIKFEPSSGGDLEVTTHSFNCRGEFFYCNTTKLFNETKLFNESEYVDNKTIILPCRIKQIINMWQEVGRAMYAPPIEGNITCKSNITGLLLTRDGGENSTEEVFRPGGGNMKDNWRSELYKYKVVEIKPLGVAPTKCKRKVVGRRRRRRAVGLGAVLLGFLGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLpRAPEAQQHLLQLTVWGIKQLQTRVLAIERYLevQQLLGLWGCSGKLICCTAVPWNSSWSNKSLGDIWDNMTWMQWDREISNYTNTIYRLLEDSQfQQEiNEKDLLALDSEQ ID NO: 27 (repaired and stabilized DS_sC4_SOSIP (HIV170686))MGNLWVTVYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDqSLkPCVKLTPLCVTLNCRNVRNVEMKNCSFNATTVVrDRKQKVHALFYRLDIVPLDENNSSYRLINCNTSAcTQiCPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTIKFAPHSGGDLEITTHsFNCRGEFFYCNTSNLFNESNIERNDSIITLPCRIKQIINMWQEVGRcmYAPPIAGNITCRSNITGLLLTRDGGSNNNDTETFRPGGGDMRNNWRSELYKYKVVEVKPLGVAPTECKRRVVERRRRRRAVGIGAVFLGILGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLpRAPEAQQHMLQLTVWGIKQLQTRVLAIERYLevQQLLGLWGCSGKLICCTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQfQQEiSEQ ID NO: 28 (Stabilized ConC_SOSIP.v3 (HIV170654))NLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTEMKNCSFNTTTEIRDKKQKEYALFYRLDIVPLNENSSEYRLINCNTSTITQICPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNVKTIIVHLNESVEIVCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFPNKTIKFQPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTKCKRRVVERRRRRRAVGIGAVFLGFLGAAGSTMGAASNTLTVQARQLLSGIVQQQSNLLRAPEAQQHMLQLTVWGFKQLQARVLAIERYLEVQQLLGIWGCSGKLICCTAVPWNSSWSNKSQEDIWDNMTWMQWDREISNYTDTIYRLLEESQFQQEINEKDLLALD SEQ ID NO: 29 (Stabilized BG505_SOSIP.v2 (HIV171814))AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAcTQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQcMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARNLLSGIVQQQSNLpRAPEAQQHLLKLTVWGIKQLQARVLAVERYLevQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQfQQEiNEvDLLALDSEQ ID NO: 30 (Repaired and stabilized C97ZA_SOSIP.v2 (HIV171810))NlWVTVYYGVPVWkDAKTTLFCASDaKAYDREVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNATFKNNVTNDMNKEIRNCSFNTTTEIRDKKQkvYALFYRlDIVqLKENRNNSNNSEYrLINCNtSTcTQiCPKVtFDPIPIHYCAPAGYAILKCNNKTFnGtGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEKEIIIRSENLTDNVKTIIVHLNKSVEInCTRPNNNTRKSiRIGPGQTFYATGDIIGDIRQAYCNISGSKWNETLKRVKEKLrEhfNNNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTTRLFNNNATEnETITLPCRIKQIINMWQeVGRcMYAPPIAGNITCKSNITGLLLtRDGGEDNKTEEIFRPGGGNMKDNWRSELYKYKVvEiKPLGIAPTkcKRRnVtRrrrrRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARQLLSgIVQQQSNLpRApEAQQHMLkLTVWGIKQLQaRVLAIERYLevQQLLGIWGCSGKLICcTNVPWNSSWSNKSQTDIWNNMTWMEWDREISNYTDTIYRLLEDSQfQQEiNEvDLLAnDSEQ ID NO: 31 (Repaired and stabilized Du422_SOSIP.v1 (HIV171812))NLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCKNVNISANANATATLNSSMNGEIKNCSFNTTTELRDKKQKVYALFYKPDVVPLNGGEHNETGEYILINCNSSTcTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNIKTIIVHLNKSVEINCTRPNNNTRKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQVKEKLREHYNKTIKFEPSSGGDLEVTTHSFNCRGEFFYCNTTKLFNETKLFNESEYVDNKTIILPCRIKQIINMWQEVGRcMYAPPIEGNITCKSNITGLLLTRDGGENSTEEVFRPGGGNMKDNWRSELYKYKVVEIKPLGVAPTKCKRKVVGRRRRRRAVGLGAVLLGFLGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLpRAPEAQQHLLQLTVWGIKQLQTRVLAIERYLevQQLLGLWGCSGKLICCTAVPWNSSWSNKSLGDIWDNMTWMQWDREISNYTNTIYRLLEDSQfQQEiNEvDLLALDSEQ ID NO: 32 (Stabilized and repaired sC4_SOSIP.v4)MGNLWVTVYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDqSLkPCVKLTPLCVTLNCRNVRNVEMKNCSFNATTVVrDRKQKVHALFYRLDIVPLDENNSSYRLINCNTSAcTQiCPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNIVCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTIKFAPHSGGDLEITTHsFNCRGEFFYCNTSNLFNESNIERNDSIITLPCRIKQIINMWQEVGRcmYAPPIAGNITCRSNITGLLLTRDGGSNNNDTETFRPGGGDMRNNWRSELYKYKVVEVKPLGVAPTECKRRnVtRRRRRRAVGIGAVFLGILGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLpRAPEAQQHMLQLTVWGIKQLQTRVLAIERYLeDQQLLGLWGCSGKLICCTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGETYRLLEESQfQQEiSEQ ID NO: 33 (example of a signal sequence (e.g. used for DS_sC4_SOSIPvariants) MRVRGMLRNWQQWWIWSSLGFWMLMIYSVSEQ ID NO: 34 (example of a signal sequence (e.g. used for BG505_SOSIPvariants) MRVMGIQRNCQHLFRWGTMILGMIIICSASEQ ID NO: 35 (Stabilized and repaired ZM233M_SOSIP (HIV172520))SLWVTVYYGVPVWREAKTTLFCASDAKAYETEvHnVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDVISlWDQSLKPCVKLTPLCVTLnCSTvNNTHNISeEMKnCSFNMTTELRDKKRKVNaLFYKLDiVPLTNSSNTTNYRLInCNTSTITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRsENLTDNVKIIIVQLNETINITCTRPNNNTRKSIRIGPGQtFYATGEIiGNIREAHCNISeSKWNKTLERVRkKLKEHFPNKTIEFEPSSGGDLEITTHSFNCGGEFFYCNTSGLFNStyNGTsTSNiTLPCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLLLTRDGGENSSnTTETFRPgGGDMKdNWRSELYKYKVVEIKPLGIAPTEcKRRVVERRRRRRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLpKApEAQQHMLQLTVWGIKQLQARVLAIERYLevQQLLGLWGCSGKLICcTNVPWNsSWSNKSKNDIWDNMTWMQWDREISNyTDTIYRLLEDSQfQQEiNEvDLLALDSEQ ID NO: 36 (Stabilized and repaired CN97001_SOSIP (HIV172523))nGNLWVTVYYGVPVWKEAkTTLFCASDAKAYDTEVHNVWATHACVPtDPNPQEMVLENVTENFNMWKNdMVdQMhEDVISLWDQSLKPCVKLTPLCVTLnCtNVSSNSNDTYHETYHESMKEMKNCSFNATTeVRDRKQTVYALFYRLDIVPLnKKNnSENSSEYYRLINCNTSAITQACPKVTFDPIPIHYCaPAGYAILKCNDKtFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENLTNNVKTIIVHLNQSVEIVCTRPGNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEDKWNETLQRVSKKLAEHFpNKTIKFASSSGGDLEVTTHSFNCRGEFFYCNTSGLFNGTYTPNGTKSNSSSIITIPCRIKQIINMWQEVGRAMYAPPIKGNITCKSNITGLLLVRDGGTEnNDTETFRPGGGDMRdNWRSELYKYKVVEIKPLGVAPTTcKRRVVERRRRRRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLpRApEAQQHLLQLTVWGIKQLQTRVLAIERYLevQQLLGIWGCSGKLICcTAVPWNSSWSNKSQEDIWDNMTWMQWDREISNYTDTIYRLLEESQfQQEiNEvDLLALDSEQ ID NO: 37 (Stabilized and repaired ZM246F_SOSIP (HIV172526))NLWVTVYYGVPVWKEAKTTLFCASDAKAYDkEVHNVWATHACVPTDPNPQEMFLENVTENFNMWKNDMVEQMHeDIISLWdQSLKPCVKLTPLCVTLNCSDVINSTGeMKNCSFnVTTELRDRKQKeHALFYRLDIVPLDENDNSSKDYRLINCNTSTITQACPKVTFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENLTDNVKTIIVQLKEPVeINCTRPNNNtRQSIRIGPGQTFFATGDIIGDIREAHCNISeTKWnETLQQVGkKLAKYFNNKTIKFTQHSGGDLEITTHSFNCRGEFFYCNTSQLFNSTYNETGSINGTGNSTItLPCRIKQIINMWQGVGQAMYAPPIAGNItCRSnITGLLLTRDGGINKSEEIFRPGGGNMKDNWRSELYKYKVVEIKPLGVAPTKcKRRVVERRRRRRAVVGLGAVFLGFLGAAGSTMGAASnTLTVQARQLLSGIVQQQNNLpRApEAQQHMLQLTVWGIKQLQARVLAIERYLevQQLLGIWGCSGKLICcTAVPWNSSWSNKSQEDIWDNMTWMQWDREISNYTNTIYRLLEDSQfQQEiNEvDLLALDSEQ ID NO: 38 (Stabilized and repaired ADM30337.1_SOSIP (HIV172517))VGNLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWAThACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDVISLWDQSLKPCVKLTPLCVTLNCTNVNNNMTNVSINNNMTGEITNCSFNITTELRDKRRQVYALFYRLDIVPLDSNSSEYRLINCNTSTVTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFnGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEKEIIIRSENLTNNAKTIIVHLNESIEINCTRPNNNTRKSVRIGPGQtFYATGDIIGDIRQAHCNISESKWNETLQKVGKKLQEHFPNKTIKFAPPSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYMHNGTTGNSSGtITLPCKIKQIINMWQEVGRAMYAPPIAGNITCESNITGLLLTRDGGTTNDTSETFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTGCKRRVVERRRRRRAVGIGAVLLGFLGAAGSTMGAASnTLTVQARQlLSGIVQQQSNLpRAPEAQQHMLQLTVWGIKQLQARVLAIERYLevQQLLGIWGCSGKlICCTNVPWNSSWSNRTQEdIWDNMTWMQWDREINNYTNTIYSLLEESQfQQEiNEvDLLALDSEQ ID NO: 39 (example of a signal sequence (e.g. used for ZM233M))MRVRGIMRNWQQWWIWGSLGFWMLIICNVnGSEQ ID NO: 40 (example of a signal sequence (e.g. used for CN97001))MRVTGIRKNYRHLWRWGTMLLGMLMISSASEQ ID NO: 41 (example of a signal sequence (e.g. used for ZM246F))MRVMGILRNCQQWWIWSILGFLMIYSVIGSEQ ID NO: 42 (example of a signal sequence (e.g. used for ADM30337.1))MRVRGILRNYPQWWIWGILGFWMLMNCNGSEQ ID NO: 43 (example of a signal sequence (e.g. used for C97ZA))MRVRGIPRNWPQWWMWGILGFWMIIICRVVGSEQ ID NO: 44 (full length ConC_SOSIP (including signal sequence, in italics))MRVRGILRNWQQWWIWGILGFWMLMICNVVGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTNNNNMKEEMKNCSFNTTTEIRDKKQKEYALFYRLDIVPLNENSSEYRLINCNTSTITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTDNAKTIIVHLNESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFPNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTKCKRRVVERekRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAPEAQQHMLQLTVWGIKQLQARVLAIERYLKDQQLLGIWGCSGKLICCTAVPWNSSWSNKSQEDIWDNMTWMQWDREISNYTDTIYRLLEESQNQQEKNEKDLLALDSWNNLWNWFDITNWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVRQGYSPLSFQTLTPNPRGPDRLGRIEEEGGEQDRDRSIRLVSGFLALAWDDLRSLCLFSYHRLRDFILIAARAVELLGRSSLRGLQRGWEALKYLGSLVQYWGLELKKSAISLLDTIAIAVAEGTDRIIELIQRICRAIRNIPRRIRQGFEAALL

REFERENCES

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It is claimed:
 1. An isolated nucleic acid molecule encoding arecombinant human immunodeficiency virus (HIV) envelope (Env) protein,wherein the amino acid at position 658 of the Env protein is selectedfrom the group consisting of Val, Ile, Phe, Met, and Ala; wherein thenumbering of the position is according to the numbering in gp160 ofHIV-1 isolate HXB2 as shown in SEQ ID NO:
 1. 2. The isolated nucleicacid molecule of claim 1, wherein the amino acid at position 658 is Val.3. The isolated nucleic acid molecule of claim 1, wherein the amino acidat position 658 is Ile.
 4. The isolated nucleic acid molecule of claim1, wherein the HIV Env protein is a clade A, B, or C HIV-1 Env protein.5. The isolated nucleic acid molecule of claim 1, wherein the HIV Envprotein further comprises Cys at positions 501 and 605 or Pro atposition 559, or Cys at positions 501 and 605 and Pro at position 559.6. The isolated nucleic acid molecule of claim 1, wherein the HIV Envprotein further comprises a replacement of the furin cleavage sequenceat positions 508-511 by RRRRRR (SEQ ID NO: 10).
 7. The isolated nucleicacid molecule of claim 1, wherein the HIV Env protein is a gp140 orgp160 protein.
 8. The isolated nucleic acid molecule of claim 1, whereinthe isolated nucleic acid molecule is an RNA molecule.
 9. A vectorcomprising the isolated nucleic acid molecule of claim 1 operably linkedto a promoter.
 10. The vector of claim 9, which is an adenovirus vector.11. A host cell comprising the isolated nucleic acid molecule ofclaim
 1. 12. A method of producing a recombinant HIV Env protein,comprising growing the host cell of claim 11 under conditions suitablefor production of the recombinant HIV Env protein.
 13. A pharmaceuticalcomposition comprising the isolated nucleic acid molecule of claim 1,and a pharmaceutically acceptable carrier.
 14. A pharmaceuticalcomposition comprising the isolated nucleic acid molecule of claim 8,and a pharmaceutically acceptable carrier.
 15. A pharmaceuticalcomposition comprising the vector of claim 9, and a pharmaceuticallyacceptable carrier.
 16. A pharmaceutical composition comprising thevector of claim 10, and a pharmaceutically acceptable carrier.
 17. Theisolated nucleic acid molecule of claim 1, wherein the HIV Env proteinfurther comprises one or more of the following amino acid residues: (i)Phe, Leu, Met, or Trp, at position 651; (ii) Phe, Ile, Met, or Trp, atposition 655; (iii) Asn or Gln, at position 535; (iv) Val, Ile or Ala atposition 589; (v) Phe or Trp, at position 573; (vi) Ile at position 204;(vii) Phe, Met, or Ile, at position 647; (viii) Gln, Glu, Ile, Met, Val,Trp, or Phe, at position 588; (ix) Lys at position 64 or Arg at position66 or Lys at position 64 and Arg at position 66; (x) Trp at position316; (xi) Cys at both positions 201 and 433; (xii) Pro at position 556or 558 or at both positions 556 and 558; (xiii) replacement of the loopat amino acid positions 548-568 (HR1-loop) by a loop having an aminoacid sequence selected form the group consisting of SEQ ID NOs 12-17;(xiv) Gly at position 568, or Gly at position 569, or Gly at position636, or Gly at both positions 568 and 636, or Gly at both positions 569and 636; (xv) Tyr at position 302, or Arg at position 519, or Arg atposition 520, or Tyr at position 302 and Arg at position 519, or Tyr atposition 302 and Arg at position 520, or Tyr at position 302 and Arg atboth positions 519 and 520; and/or (xvi) Cys at positions 501 and 605,or Pro at position 559, or Cys at positions 501 and 605 and Pro atposition 559, wherein the numbering of the positions is according to thenumbering in gp160 of HIV-1 isolate HXB2 as shown in SEQ ID NO:
 1. 18.The isolated nucleic acid molecule of claim 17, wherein the HIV Envprotein comprises at least two of the amino acid residues of (i) to(vii).